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Antral gastrin regulation
JOURNAL
OF SURGICAL
RESEARCH
20,509-514 (1976)
Antral
Gastrin
Regulation’
BARRY A. LEVINE, M.D., A. GERSON GREENBURG, M.D., Ph.D., GERALD W. PESKIN, M.D., AND RICHARD P. SAIK, M.D.
Departments of Surgery, Veterans Hospital, San Diego, Califbrnia 92161; and the University of California, San Diego, California 92103 Submitted for publication December I I, 1976
recorded with a Radiometer pH electrode fluoroscopically placed in the antrum. A second series of patients (18 normal and 12 duodenal ulcer) was studied in the same manner using only pH (0.1 N HC1 and 0.1 N NaHCO, buffered saline via a No. 10 feeding tube) as an antral stimulant. On different occasions, pH values were varied from acid to alkali and alkali to acid with serum gastrin levels being measured on peripheral blood samples. All studies were begun only after a suitable control period and 2 serum samples were drawn, 15 and 30 min after each pH manipulation. Antral pH was monitored and additional solution was added as needed to maintain a constant. B. Dogs. Vagally intact antral pouches were constructed acutely in 11 anesthesized dogs by dividing the antrum between clamps at the pylorus and from the fundus. Pouches were exteriorized onto the abdominal wall along with construction of the Heidenhain pouch in four chronic dogs who were each studied 2-3 times after a 3 week recovery period. In both groups of animals, antral pH was varied from acid to alkaline and alkaline to acid with solutions of HCl and NaHCO, without distending the pouches. Confirmation of the antral mucosal pH was obtained METHODS by using a pH meter and electrode (RadioStudies were performed as follows: meter). Samples for serum gastrin were A. Human. A series of 12 patients (6 drawn from a cannulated antral draining vein normal and 6 duodenal ulcer) were fed a test in the acute animals and from a peripheral meal of 180 g of hamburger and their pe- vein in the chronic awake animals. All studies ripheral serum gastrin response was followed were started only after a suitable control peevery 15 min along with their antral pH, riod. Samples were drawn at 15 and 30 min ‘Supported by USPHS Grant AM15986-04 and VA intervals following pH manipulation. Results Grants #7001,3271, and7691 are expressed as percentage of basal, each
The relationship of antral gastrin release to various intraluminal stimuli has been confused by many conflicting reports. McGuigan and Trudeau [15], Byrnes et al. [2], and Reeder et al. [16] have all reported significant elevations of serum gastrin in patients with duodenal ulcer compared to normals, both in the fasting state and in response to feeding. However, Wyllie et al. [20] and Stern and Walsh [17] have reported no differences in gastrin levels between normal and duodenal ulcer patients. In these studies, only serum gastrin as a function of time was measured. A few, such as Wyllie et al. [20] attempted to measure intraluminal pH as well but encountered difficulty due to residue and masticated food. Some measure of antral acidity is paramount in any study of gastrin related physiology. While Woodward et al. [19], among others, have demonstrated that intraluminal acid seems to depress a stimulated level of antral gastrin release, newer studies have failed to confirm stimulation or release of gastrin by alkalinity alone [8, 11, 171. This study examines the effect of pH alone and with a protein meal on gastrin release in normal and ulcer bearing man and in dogs.
509 Copyright @1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
510
JOURNAL
OF SURGICAL
RESEARCH:
animal serving as his own control. Significance was established by the paired (t) test. Integrated gastrin output following the meal was also calculated and expressed in picogram-minutes/ml. Significance was established by Student’s t test. In addition to sampling serum gastrin levels, biopsies of antral mucosa were taken from the pouches 30 min after each pH change, and from patients with and without ulcer disease at operation. This tissue provided material for the study of G-cell population by an immunofluorescence technique. Our gastrin assay is performed using antigastrin serum prepared in white New Zealand rabbits to synthetic human gastrin and synthetic human gastrin labeled with lssI (Imperial Chemical Co., Ltd.). Titers at l/500 gave 71% binding and a nonspecific binding of only 0.04%. Normal assays were run with antiserum dilutions of l/75,000 giving 3 1% binding. Antibody to synthetic human gastrin I is prepared by ammonium sulfate precipitation at a 50% saturation. This raw antiserum is diluted I:20 with 0.1 M borate buffer (pH 8.6) and an equal volume of saturated ammonium sulfate (0°C) is added slowly. Precipitation proceeds for 30 min at 0°C when the globulin rich precipitate is separated by tentrifugation (3000 rpm for 30 min). The precipitate is dissolved in borate buffer and dialyzed to remove the ammonium sulfate and then put through diethyl aminoethyl cellulose (DEAE) chromatography. The IgG fraction which voids is concentrated to 10 mg/ml using negative pressure dialysis. This IgG fraction is fluoresceinated by dialysis using 0.025 M carbonate buffer (pH 9.6) with fluorescein isothiocyanate (FITC) dissolved at a concentration of 0.1 mg/ml. A sufficient volume of FITC containing carbonate buffer is used to provide 1 mg of FITC/lO mg of IgG. The fluoresceination process takes 8-24 hr of dialysis at 4” C. The conjugated protein is then freed of unbound FITC by further dialysis in PBS. A 0.05-0.1 M fraction is ideal for tissue staining. Antral mucosal tissue sections fixed in
VOL. 20, NO. 6, JUNE
ct,/0
1976
____________ __----____--I5
30 45 TIME-MINUTES
60
75
FIG. I. Integrated gastrin response to 180 g protein meal in normal and ulcer patients (12 patients, expressed as percentage of fasting serum gastrin over 75 min).
10% formalin and embedded in paraffin are washed to remove the fixative. The moist tissue section is transferred into a staining chamber. Twenty microliters of the fluorescein reagent are pipetted into the level moist chamber. The duration of staining lasts a minimum of 12 hr and may be extended for as long as 24 hr to enhance weak reactions. After washing, the sections are covered by mounting with 0.01 M Tris buffered glycerin (1.9), pH 9.6. Color photomicrographs (35 mm) were taken through the Leitz fluorescence microscope. Photomicrographs are taken every 200-300 pm. Gastrin cell density is obtained by counting cells per high power field (HPF) for all photomicrographs taken of any specimen. Independent observations by three unbiased viewers are classified into high (6 or greater G-cell/HPF), medium (3-5/HPF), low (<2/HPF) and results are expressed as the percentage of total observations in each category. Analysis of variance is used to determine significance.
FIG. 2. Integrated gastrin response to a meal in 6 normal patients, expressed as percentage of fasting gastrin.
LEVINE
ET AL.: ANTRAL
GASTRIN
REGULATION
511
r 150 125 100 75 50
0 30 60 0 30 60 0 30 60 TIME-MINUTES FIG. 3. Integrated gastrin response to a meal in 6 duodenal ulcer patients expressed as percentage of fasting gastrin.
RESULTS Fasting gastrin serum levels were similar in normal and duodenal ulcer patients (142 A 50 vs 128 * 38 pg/ml). The integrated gastrin output (Fig. 1) following a protein meal, however, was significantly greater in the duodenal ulcer group. Individual gastrin responses, expressed as percentage of basal, are shown in Figs. 2 and 3 (normal and ulcer patients respectively). Intragastric pH (pH 1 to 8) did not correlate with any significant changes in peripheral serum gastrin in either normal or ulcer patients compared to basal levels (Fig. 4). Normals demonstrated some acid inhibition by regression curve analysis, but the correlation was not statistically significant. Significant differences could not be appreciated if the antrum was topically perfused from low pH (1) to high pH (8) or perfusing first from high pH (8) to low pH (1) (Fig. 5). The tendency for acid inhibition of gastric release in normal but not ulcer patients, noted in Fig. 4, becomes more ap-
FIG. 4. Correlation of intragastric pH and serum gastrin (expressed as percentage of fasting gastrin) in normal and ulcer patients in response to a meal.
25
FIG. 5. Effect of intragastric lavage with solutions of different pH alone in 30 normal and ulcer patients. Serum gastrin expressed as percentage of basal gastrin.
parent by examining the number of occasions in which inhibition of gastrin occurred in each group at an acid pH (<2.0). On 17 out of 28 occasions, acid inhibition was noted in normals, but in only 5 out of 15 in the ulcer group was there a decrease in gastrin (P = 0.043) (Table 1). In the animal experiments, no differences in serum gastrin levels to either the acid or alkaline antral challenge were evident in the acute dog model sampling from an antral vein (182 vs 170 pg/ml) (Fig. 6), or in the chronic dogs, sampling peripherally (82 vs 9 1 pg/ml) (Fig. 7). Finally, immunofluorescent studies of human antral G-cell density from duodenal ulcer and normal patients failed to elucidate any differences between the two groups, both containing an abundance of G-cells. However, in the dog experiments, there was a decrease in total G-cell immunofluorescence in the mucosa perfused in an alkaline milieu compared to acid (Table 2). DISCUSSION Many aspects of antral gastrin regulation and release in normal and duodenal ulcer patients have been debated in recent years. This study attempted to examine three areas concerned with that regulation: (1) Quantitative differences between normal and duodenal ulcer patients in antral gastrin release as stimulated by a protein meal; (2) differences in response of the antrum, as measured by serum gastrin, to acid and al-
512
JOURNAL
OF SURGICAL
RESEARCH:
VOL. 20, NO. 6, JUNE
1976
TABLE I Effect of Lowering Intragastric pH to I 2 on Serum Gastrin Compared to Basal Levels in Normal and Ulcer Patients Acid inhibition ofgastrin
% Observations with gastrin basal *Significant
at acid pH (52.0)
Normal
Duodenal ulcer
60.7*(17/28) 39.3 (I l/28)
33.3 ( 5/M) 66.6*(10/15)
at PcO.05.
kaline stimuli alone; and (3) histological examination of stimulated antra to study correlation of serum gastrin to degranulation of the gastrin cell (G-cell) in response to stimuli. This study supports the concept that gastrin release following a protein meal is significantly greater in duodenal ulcer patients despite lack of significant differences in basal levels. Whether, in fact, this has importance to the pathogenesis of the peptic ulcer syndrome is unresolved, for despite increased gastrin levels, acid output may not correlate. The increased food stimulated gastrin levels in duodenal ulcer patients may point to a different level of antral G-cell responsiveness in the ulcer group since a difference in G-cell density could not be demonstrated. Most previous studies have not used pH change alone, without other agents, as an antral stimulus. Konturek et al. [12] used a peptone meal with pH adjustments l-5.5; Jackson et al. [lo] used acetylcholine of pH 1.6 and 7.0 and Elwin and Andersson [5] used acetylcholine, choline, glycine and ethanol at various pH values. Csendes et al. [3] and
Debas et al. [4] used insulin challenge and distention of a fundic pouch in conjunction with alteration of pH within a vagally innervated pouch. Walsh et al. [18] has also used an amino acid meal as a stimulus while varying intragastric pH from 2.5 to 5.5. All these studies have shown that acidification of the antrum will decrease the gastrin response to a known stimulus. There are now several studies which examine pH alteration of the antral mucosa and gastrin release, without other concomitant stimulation. Thus, Kline et al. [l l] has used NaHCO, solutions to raise the pH to 7.0 from a control of 2.0 without any increase in serum gastrin levels noted. Higgs et al. [8] studied the effect of various calcium and noncalcium containing antacids intragastrically on serum gastrin, finding no discernible significant changes. This present study supports these observations and demonstrates no significant change in serum gastrin levels due to topical pH manipulations of the antral mucosa except for inhibition in normal patients at an acid pH. Taken together with the other previously mentioned studies, these observations sug-
PH bhd I30
FIG. 6. Acute dogs; I = SD. Effect of topical pH changes in the antral pouch in anesthesized dogs on antral vein gastrin levels.
FIG. 7. Chronic dogs; I = SD. Effect of topical pH change in exteriorized antral pouch in chronic, awake dogs on peripheral serum gastrin levels.
LEVINE ET AL.: ANTRAL
513
GASTRIN REGULATION
TABLE 2 Topical pH Effect on Antral G-Cell Density in Dogs’ PH
N
Low (2 cells/HPF)
I 2 3 4 5 6
35 36 24 29 16 6
IO 20.4 37.5 68.9* 72.9* 83.3*
Medium (3-5 cells/HPF) I6 35.2 19.4 13.8 14.6 5.5
High (6 cells/HPF) 73.9* 44.4 43.0 17.2 12.5 II.1
‘Categorization of the percentage of observations of cell density by number per high power field after 30 min at each pH. *Significant at P = 0.04.
gest that perhaps antral pH is not a major that increased gastrin release in the absence stimulant of basal antral gastrin release but of acid occurs only after some period of time rather a modifier of other stimuli, with al- during which G-cell hyperplasia occurs. Thus, we have demonstrated that the Gkalinity acting in a permissive fashion. In cells of duodenal ulcer patients respond more normal individuals, acid inhibition does seem to decrease serum gastrin as a negative feed- readily to a protein milieu than normal back mechanism apparently lacking in ulcer patients and appear to lack an acid feedback patients. Lack of this mechanism plus the inhibition. The antral G-cell, however, does increased gastrin response to a meal, indi- not appear to stimulate in response to acute cates that there is a difference in G-cell regu- elevations in pH alone over the total range of clinical values. This lack of gastrin response lation in the ulcer patient. The immunofluorescence studies were un- to alkaline stimulation occurs both in normal dertaken to correlate any observed fluctua- and duodenal ulcer patients as well as in the tions in serum gastrin levels with his- dog model and casts doubt on the role of altochemical evidence of G-cell change. The kali as a primary stimulant of G-cell funcdog antral biopsies at various pH indicate tion. that G-cell discharge is prevented in an acid SUMMARY environment demonstrated by a greater immunofluorescence staining (i.e., more GExperiments have been carried out in hucells/HPF at low pH). This also supports the mans and animal subjects in an effort to concept of acid as a negative feedback regu- further delineate antral mechanisms of lator. Despite the failure of the alkaline gastrin release. Protein meals and variation milieu to produce a significantly elevated of antral pH with buffered saline has been gastrin level, G-cell discharge may actually employed. occur as evidenced by decreased G-cell stainResults show that: ing (i.e., fewer G-cells/HPF). Similar antral 1. Fasting gastrin levels in duodenal ulcer pH manipulation and biopsy could not be patients and normals are the same. done in humans. The short-term pH changes, 2. Antral G-cells of duodenal ulcer subas in this study, would be expected to affect jects respond more readily than normals to a only change in cell size or the state of granu- protein meal stimulus. lation. Thus, while this study demonstrates 3. Variation in antral pH over the total fewer G-cells at more alkaline pH values, range of clinical values does not significantly chronic pH changes, such as achlorhydria or stimulate basal gastrin levels. Significant a constant alkaline stimulus, have been acid inhibition occurred in normal patients demonstrated to result in increased G-cell only. density in the antrum [13, 141. This suggests 4. G-cell degranulation seems to occur in
514
JOURNAL
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an alkaline milieu or be blocked by acidity, i.e., more G-cells are seenat an acid pH. 5. The negative feedblock control over antral gastrin release appears to be less efficient in ulcer patients. ACKNOWLEDGMENTS The authors thank Stephen Forbes and Laura Terlizzi for their technical assistance in this study,
REFERENCES I. Becker, H. D., Reeder, D. D., and Thompson, J. C. Effect of atropine on basal and food-stimulated serum gastrin levels in man. Surgery 75:701, 1974. 2. Byrnes, D. J., Young, J. D., Chisholm, D., and Lazarus, L. Serum gastrin in patients with peptic ulceration. Bn’f. Med. J. 2:626, 1970. 3. Csendes, A., Walsh, J. H., and Grossman, M. I. Effects of atropine and of antral acidification on gastrin release and acid secretion in response to insulin and feeding in dogs. Gasrroenferology 63:257, 1972. 4. Debas, H. T., Walsh, J. H., and Grossman, M. I. Evidence for oxyntopyloric reflex for release of antralgastrin. Gusfroenterology68:687, 1975. 5. Elwin, C. E., and Andersson, S. Influence of pH on release of gastrin by chemical agents. Acta Physiol. Stand. 68~44, 1966. 6. Farrell, R. L., Castell, D. O., and McQuigan, J. E. Measurement and comparisons of lower esophageal sphincter pressure and serum gastrin levels in patients with gastroesophageal reflux. Gastroenrerology67:415, 1974. 7. Hansky, J., Korman, M. G., Cowley, D. J., and Baron, J. H. Serum gastrin in duodenal ulcer. II. Cur 12:959, 1971. 8. Higgs, R. H., Smyth, R. D., and Castell, D. 0. Gastrin alkalinization: Effect on LES pressure and serum gastrin. N. Engl. J. Med. 291:486, 1974. 9. Isenberg, J. I., Walsh, J. H., Best, W. R., and Grossman, M. I. Effect of graded doses of
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1976
pentagastrin on gastric acid secretion in duodenal ulcer and non-duodenal ulcer subjects. Gastroenteroiogy 62~764, 1972. IO. Jackson, B. M., Reeder, D. D., and Thompson, J. C. Dynamic characteristics of gastrin release. Amer. J. Surg.123:137,1972.
I1
Kline, M. M., McCallem, R. W., Curry, N., and Sturdevant, R. A. Effect of gastric alkalinization of LES pressure and serum gastrin. Gastroenterology 68:1137,1975. 12 Konturek, S. J., Biernat, J., and Oleksy, J. Serum gastrin and gastric acid responses to meals at various pH levels in man. Gut 15:526, 1974. 13. Korman, M. G., Hansky, J., and Stickland, R. F., Progressive increase in the functional G-cell mass with age in atrophic gastritis. Gut 14:549,1973. 14. Lehy, T., Voillemot, N., Dubrasquet, M., and Dufougeray, F., Gastrin cell hyperplasia in rats with chronic antral stimulation. Gastroenterology 68:71, 1975. 15. McGuigan, J. E., and Trudeau, B. M. Differences in rats of gastrin release in normal persons and patients with duodenal ulcer disease. N. Engl. J. Med. 288:64, 1973. 16. Reeder, D. D., Jackson, B. M., Ban, J., Davidson, W., and Thompson, J. C., Effect of food on serum gastrin concentration in duodenal ulcer and control patients. Surg. Forum 21:290, 1970. 17. Stern, D. H., and Walsh, J. H. Gastrin release in postoperative ulcer patients: Evidence for release of duodenal gastrin. Gastroenrerology 64:363, 1973. 18. Walsh, J. H., Richardson, C. T., and Fordtran, J. S. pH dependence of acid secretion and gastrin release in normal and ulcer subjects. J. Clin. Invest. 55:462, 1975. 19. Woodward, E. R., Lyon, E. S., Landor, J., and Dragstedt, L. R. Physiology of the gastric antrum: Experimental studies on isolated antrum pouches in dogs. Gastroenrerology27:766, 1954. 20. Wyllie J., Boulos, P. B., Lewin, M. L., Stagg, B. H., and Clark, C. G., Plasma gastrin and acid secretion in man following stimulation by food, meat extract, and insulin. Gut 13:887, 1972.
OF SURGICAL
RESEARCH
20,509-514 (1976)
Antral
Gastrin
Regulation’
BARRY A. LEVINE, M.D., A. GERSON GREENBURG, M.D., Ph.D., GERALD W. PESKIN, M.D., AND RICHARD P. SAIK, M.D.
Departments of Surgery, Veterans Hospital, San Diego, Califbrnia 92161; and the University of California, San Diego, California 92103 Submitted for publication December I I, 1976
recorded with a Radiometer pH electrode fluoroscopically placed in the antrum. A second series of patients (18 normal and 12 duodenal ulcer) was studied in the same manner using only pH (0.1 N HC1 and 0.1 N NaHCO, buffered saline via a No. 10 feeding tube) as an antral stimulant. On different occasions, pH values were varied from acid to alkali and alkali to acid with serum gastrin levels being measured on peripheral blood samples. All studies were begun only after a suitable control period and 2 serum samples were drawn, 15 and 30 min after each pH manipulation. Antral pH was monitored and additional solution was added as needed to maintain a constant. B. Dogs. Vagally intact antral pouches were constructed acutely in 11 anesthesized dogs by dividing the antrum between clamps at the pylorus and from the fundus. Pouches were exteriorized onto the abdominal wall along with construction of the Heidenhain pouch in four chronic dogs who were each studied 2-3 times after a 3 week recovery period. In both groups of animals, antral pH was varied from acid to alkaline and alkaline to acid with solutions of HCl and NaHCO, without distending the pouches. Confirmation of the antral mucosal pH was obtained METHODS by using a pH meter and electrode (RadioStudies were performed as follows: meter). Samples for serum gastrin were A. Human. A series of 12 patients (6 drawn from a cannulated antral draining vein normal and 6 duodenal ulcer) were fed a test in the acute animals and from a peripheral meal of 180 g of hamburger and their pe- vein in the chronic awake animals. All studies ripheral serum gastrin response was followed were started only after a suitable control peevery 15 min along with their antral pH, riod. Samples were drawn at 15 and 30 min ‘Supported by USPHS Grant AM15986-04 and VA intervals following pH manipulation. Results Grants #7001,3271, and7691 are expressed as percentage of basal, each
The relationship of antral gastrin release to various intraluminal stimuli has been confused by many conflicting reports. McGuigan and Trudeau [15], Byrnes et al. [2], and Reeder et al. [16] have all reported significant elevations of serum gastrin in patients with duodenal ulcer compared to normals, both in the fasting state and in response to feeding. However, Wyllie et al. [20] and Stern and Walsh [17] have reported no differences in gastrin levels between normal and duodenal ulcer patients. In these studies, only serum gastrin as a function of time was measured. A few, such as Wyllie et al. [20] attempted to measure intraluminal pH as well but encountered difficulty due to residue and masticated food. Some measure of antral acidity is paramount in any study of gastrin related physiology. While Woodward et al. [19], among others, have demonstrated that intraluminal acid seems to depress a stimulated level of antral gastrin release, newer studies have failed to confirm stimulation or release of gastrin by alkalinity alone [8, 11, 171. This study examines the effect of pH alone and with a protein meal on gastrin release in normal and ulcer bearing man and in dogs.
509 Copyright @1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
510
JOURNAL
OF SURGICAL
RESEARCH:
animal serving as his own control. Significance was established by the paired (t) test. Integrated gastrin output following the meal was also calculated and expressed in picogram-minutes/ml. Significance was established by Student’s t test. In addition to sampling serum gastrin levels, biopsies of antral mucosa were taken from the pouches 30 min after each pH change, and from patients with and without ulcer disease at operation. This tissue provided material for the study of G-cell population by an immunofluorescence technique. Our gastrin assay is performed using antigastrin serum prepared in white New Zealand rabbits to synthetic human gastrin and synthetic human gastrin labeled with lssI (Imperial Chemical Co., Ltd.). Titers at l/500 gave 71% binding and a nonspecific binding of only 0.04%. Normal assays were run with antiserum dilutions of l/75,000 giving 3 1% binding. Antibody to synthetic human gastrin I is prepared by ammonium sulfate precipitation at a 50% saturation. This raw antiserum is diluted I:20 with 0.1 M borate buffer (pH 8.6) and an equal volume of saturated ammonium sulfate (0°C) is added slowly. Precipitation proceeds for 30 min at 0°C when the globulin rich precipitate is separated by tentrifugation (3000 rpm for 30 min). The precipitate is dissolved in borate buffer and dialyzed to remove the ammonium sulfate and then put through diethyl aminoethyl cellulose (DEAE) chromatography. The IgG fraction which voids is concentrated to 10 mg/ml using negative pressure dialysis. This IgG fraction is fluoresceinated by dialysis using 0.025 M carbonate buffer (pH 9.6) with fluorescein isothiocyanate (FITC) dissolved at a concentration of 0.1 mg/ml. A sufficient volume of FITC containing carbonate buffer is used to provide 1 mg of FITC/lO mg of IgG. The fluoresceination process takes 8-24 hr of dialysis at 4” C. The conjugated protein is then freed of unbound FITC by further dialysis in PBS. A 0.05-0.1 M fraction is ideal for tissue staining. Antral mucosal tissue sections fixed in
VOL. 20, NO. 6, JUNE
ct,/0
1976
____________ __----____--I5
30 45 TIME-MINUTES
60
75
FIG. I. Integrated gastrin response to 180 g protein meal in normal and ulcer patients (12 patients, expressed as percentage of fasting serum gastrin over 75 min).
10% formalin and embedded in paraffin are washed to remove the fixative. The moist tissue section is transferred into a staining chamber. Twenty microliters of the fluorescein reagent are pipetted into the level moist chamber. The duration of staining lasts a minimum of 12 hr and may be extended for as long as 24 hr to enhance weak reactions. After washing, the sections are covered by mounting with 0.01 M Tris buffered glycerin (1.9), pH 9.6. Color photomicrographs (35 mm) were taken through the Leitz fluorescence microscope. Photomicrographs are taken every 200-300 pm. Gastrin cell density is obtained by counting cells per high power field (HPF) for all photomicrographs taken of any specimen. Independent observations by three unbiased viewers are classified into high (6 or greater G-cell/HPF), medium (3-5/HPF), low (<2/HPF) and results are expressed as the percentage of total observations in each category. Analysis of variance is used to determine significance.
FIG. 2. Integrated gastrin response to a meal in 6 normal patients, expressed as percentage of fasting gastrin.
LEVINE
ET AL.: ANTRAL
GASTRIN
REGULATION
511
r 150 125 100 75 50
0 30 60 0 30 60 0 30 60 TIME-MINUTES FIG. 3. Integrated gastrin response to a meal in 6 duodenal ulcer patients expressed as percentage of fasting gastrin.
RESULTS Fasting gastrin serum levels were similar in normal and duodenal ulcer patients (142 A 50 vs 128 * 38 pg/ml). The integrated gastrin output (Fig. 1) following a protein meal, however, was significantly greater in the duodenal ulcer group. Individual gastrin responses, expressed as percentage of basal, are shown in Figs. 2 and 3 (normal and ulcer patients respectively). Intragastric pH (pH 1 to 8) did not correlate with any significant changes in peripheral serum gastrin in either normal or ulcer patients compared to basal levels (Fig. 4). Normals demonstrated some acid inhibition by regression curve analysis, but the correlation was not statistically significant. Significant differences could not be appreciated if the antrum was topically perfused from low pH (1) to high pH (8) or perfusing first from high pH (8) to low pH (1) (Fig. 5). The tendency for acid inhibition of gastric release in normal but not ulcer patients, noted in Fig. 4, becomes more ap-
FIG. 4. Correlation of intragastric pH and serum gastrin (expressed as percentage of fasting gastrin) in normal and ulcer patients in response to a meal.
25
FIG. 5. Effect of intragastric lavage with solutions of different pH alone in 30 normal and ulcer patients. Serum gastrin expressed as percentage of basal gastrin.
parent by examining the number of occasions in which inhibition of gastrin occurred in each group at an acid pH (<2.0). On 17 out of 28 occasions, acid inhibition was noted in normals, but in only 5 out of 15 in the ulcer group was there a decrease in gastrin (P = 0.043) (Table 1). In the animal experiments, no differences in serum gastrin levels to either the acid or alkaline antral challenge were evident in the acute dog model sampling from an antral vein (182 vs 170 pg/ml) (Fig. 6), or in the chronic dogs, sampling peripherally (82 vs 9 1 pg/ml) (Fig. 7). Finally, immunofluorescent studies of human antral G-cell density from duodenal ulcer and normal patients failed to elucidate any differences between the two groups, both containing an abundance of G-cells. However, in the dog experiments, there was a decrease in total G-cell immunofluorescence in the mucosa perfused in an alkaline milieu compared to acid (Table 2). DISCUSSION Many aspects of antral gastrin regulation and release in normal and duodenal ulcer patients have been debated in recent years. This study attempted to examine three areas concerned with that regulation: (1) Quantitative differences between normal and duodenal ulcer patients in antral gastrin release as stimulated by a protein meal; (2) differences in response of the antrum, as measured by serum gastrin, to acid and al-
512
JOURNAL
OF SURGICAL
RESEARCH:
VOL. 20, NO. 6, JUNE
1976
TABLE I Effect of Lowering Intragastric pH to I 2 on Serum Gastrin Compared to Basal Levels in Normal and Ulcer Patients Acid inhibition ofgastrin
% Observations with gastrin
at acid pH (52.0)
Normal
Duodenal ulcer
60.7*(17/28) 39.3 (I l/28)
33.3 ( 5/M) 66.6*(10/15)
at PcO.05.
kaline stimuli alone; and (3) histological examination of stimulated antra to study correlation of serum gastrin to degranulation of the gastrin cell (G-cell) in response to stimuli. This study supports the concept that gastrin release following a protein meal is significantly greater in duodenal ulcer patients despite lack of significant differences in basal levels. Whether, in fact, this has importance to the pathogenesis of the peptic ulcer syndrome is unresolved, for despite increased gastrin levels, acid output may not correlate. The increased food stimulated gastrin levels in duodenal ulcer patients may point to a different level of antral G-cell responsiveness in the ulcer group since a difference in G-cell density could not be demonstrated. Most previous studies have not used pH change alone, without other agents, as an antral stimulus. Konturek et al. [12] used a peptone meal with pH adjustments l-5.5; Jackson et al. [lo] used acetylcholine of pH 1.6 and 7.0 and Elwin and Andersson [5] used acetylcholine, choline, glycine and ethanol at various pH values. Csendes et al. [3] and
Debas et al. [4] used insulin challenge and distention of a fundic pouch in conjunction with alteration of pH within a vagally innervated pouch. Walsh et al. [18] has also used an amino acid meal as a stimulus while varying intragastric pH from 2.5 to 5.5. All these studies have shown that acidification of the antrum will decrease the gastrin response to a known stimulus. There are now several studies which examine pH alteration of the antral mucosa and gastrin release, without other concomitant stimulation. Thus, Kline et al. [l l] has used NaHCO, solutions to raise the pH to 7.0 from a control of 2.0 without any increase in serum gastrin levels noted. Higgs et al. [8] studied the effect of various calcium and noncalcium containing antacids intragastrically on serum gastrin, finding no discernible significant changes. This present study supports these observations and demonstrates no significant change in serum gastrin levels due to topical pH manipulations of the antral mucosa except for inhibition in normal patients at an acid pH. Taken together with the other previously mentioned studies, these observations sug-
PH bhd I30
FIG. 6. Acute dogs; I = SD. Effect of topical pH changes in the antral pouch in anesthesized dogs on antral vein gastrin levels.
FIG. 7. Chronic dogs; I = SD. Effect of topical pH change in exteriorized antral pouch in chronic, awake dogs on peripheral serum gastrin levels.
LEVINE ET AL.: ANTRAL
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GASTRIN REGULATION
TABLE 2 Topical pH Effect on Antral G-Cell Density in Dogs’ PH
N
Low (2 cells/HPF)
I 2 3 4 5 6
35 36 24 29 16 6
IO 20.4 37.5 68.9* 72.9* 83.3*
Medium (3-5 cells/HPF) I6 35.2 19.4 13.8 14.6 5.5
High (6 cells/HPF) 73.9* 44.4 43.0 17.2 12.5 II.1
‘Categorization of the percentage of observations of cell density by number per high power field after 30 min at each pH. *Significant at P = 0.04.
gest that perhaps antral pH is not a major that increased gastrin release in the absence stimulant of basal antral gastrin release but of acid occurs only after some period of time rather a modifier of other stimuli, with al- during which G-cell hyperplasia occurs. Thus, we have demonstrated that the Gkalinity acting in a permissive fashion. In cells of duodenal ulcer patients respond more normal individuals, acid inhibition does seem to decrease serum gastrin as a negative feed- readily to a protein milieu than normal back mechanism apparently lacking in ulcer patients and appear to lack an acid feedback patients. Lack of this mechanism plus the inhibition. The antral G-cell, however, does increased gastrin response to a meal, indi- not appear to stimulate in response to acute cates that there is a difference in G-cell regu- elevations in pH alone over the total range of clinical values. This lack of gastrin response lation in the ulcer patient. The immunofluorescence studies were un- to alkaline stimulation occurs both in normal dertaken to correlate any observed fluctua- and duodenal ulcer patients as well as in the tions in serum gastrin levels with his- dog model and casts doubt on the role of altochemical evidence of G-cell change. The kali as a primary stimulant of G-cell funcdog antral biopsies at various pH indicate tion. that G-cell discharge is prevented in an acid SUMMARY environment demonstrated by a greater immunofluorescence staining (i.e., more GExperiments have been carried out in hucells/HPF at low pH). This also supports the mans and animal subjects in an effort to concept of acid as a negative feedback regu- further delineate antral mechanisms of lator. Despite the failure of the alkaline gastrin release. Protein meals and variation milieu to produce a significantly elevated of antral pH with buffered saline has been gastrin level, G-cell discharge may actually employed. occur as evidenced by decreased G-cell stainResults show that: ing (i.e., fewer G-cells/HPF). Similar antral 1. Fasting gastrin levels in duodenal ulcer pH manipulation and biopsy could not be patients and normals are the same. done in humans. The short-term pH changes, 2. Antral G-cells of duodenal ulcer subas in this study, would be expected to affect jects respond more readily than normals to a only change in cell size or the state of granu- protein meal stimulus. lation. Thus, while this study demonstrates 3. Variation in antral pH over the total fewer G-cells at more alkaline pH values, range of clinical values does not significantly chronic pH changes, such as achlorhydria or stimulate basal gastrin levels. Significant a constant alkaline stimulus, have been acid inhibition occurred in normal patients demonstrated to result in increased G-cell only. density in the antrum [13, 141. This suggests 4. G-cell degranulation seems to occur in
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an alkaline milieu or be blocked by acidity, i.e., more G-cells are seenat an acid pH. 5. The negative feedblock control over antral gastrin release appears to be less efficient in ulcer patients. ACKNOWLEDGMENTS The authors thank Stephen Forbes and Laura Terlizzi for their technical assistance in this study,
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pentagastrin on gastric acid secretion in duodenal ulcer and non-duodenal ulcer subjects. Gastroenteroiogy 62~764, 1972. IO. Jackson, B. M., Reeder, D. D., and Thompson, J. C. Dynamic characteristics of gastrin release. Amer. J. Surg.123:137,1972.
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