Suppression of Gastric Secretion and Serum Gastrin by Gastrin Antibody Hugo V. Vlllar, MD, Galveston, Texas Takaho Watayou, MD, Galveston, Texas
Robert A. D. Booth, FRCS, Galveston, Texas Phillip L. Rayford, PhD, Galveston, Texas David D. Reeder, MD, Galveston, Texas James C. Thompson, MD, Galveston, Texas
Neutralization of circulating gastrin, which would result in diminution in gastric acid secretion, might be effective in the treatment of peptic ulcer disease, especially in patients with the ZollingerEllison syndrome. A preliminary report [I] suggested that gastrin could be neutralized by the administration of antigastrin antibody (Abe) in man. Incubation of gastrin with specific antibody in vitro, followed by administration of the mixture to rats, has been shown to abolish biologic activity of gastrin (21. Inactivation of hormone by antibody in vitro is related to the dose of hormone and the concentration of antibody used [3]. In order to learn more about the mechanism of hormonal inactivation in vivo, we have studied the effects on gastric secretion and on circulating serum gastrin (as measured by radioimmunoassay) of varying doses of either normal rabbit serum or antigastrin antibody administered to dogs. Material and Methods Production of Antigastrin Antibody. Synthetic human gastrin (2 to 17 amino acids) was used as an immunogen to generate antigastrin antibody in New Zealand white rabbits. The rabbits were bled weekly, and serum was collected and tested for antibody titer. A pool of undiluted antigastrin antibody (found to bind approximately 1.7 pg synthetic human gastrin per ml by radioimmunoassay) was used for these studies. Fourteen healthy mongrel dogs were divided into three experimental groups. Group I (chronic study, single Injection)
Group la. Four dogs were prepared pouches. Three weeks after recovery,
with Heidenhain each dog received
From the Department of Surgery, The University of Texas Medical Branch. Wveston. Texas. This work was suppcfted by a grant from the National Institutes of Health (AM 15241). Reprint requests should be addressed to James C Thompson, MD, Department of Surgery. The University of Texas Medical Branch. Galveston, Texas 77550. Presented at the Sixteenth Annual Meeting of the Society for Surgery of the Alimentary Tract, San Antonio, Texas, May 20-21. 1975.
64
a single dose of either 0.001, 0.005, 0.01 or 0.03 ml/kg of Abe intravenously. Basal blood samples for serum gastrin determination were drawn daily one week before and for two weeks after injection of the antigastrin antibody. Two food studies were performed before the day of injection, and one, three, and ten days after the injection of antigastrin antibody. At each study, blood samples for gastrin were drawn before feeding a standard 400 gm meat meal and at 5, 10, 15, 30, 60, 90, 120, and 180 minutes after feeding. Gastric secretions were collected at 15 minute intervals for 180 minutes throughout the test period. Group lb. Two additional dogs without Heidenhain pouches were injected with a single dose of 0.03 and 0.07 ml/kg of antigastrin antibody. Basal blood samples for gastrin determinations were drawn before and daily after injection for four months. Group [I (chronk study, multiple Injection)
Group Ila (control). Four dogs were prepared with Heidenhain pouches. Three weeks after recovery, three of them received a daily injection of 0.03 ml/kg of normal rabbit serum for eleven days intravenously. The fourth dog received 0.07 ml/kg of normal rabbit serum for eleven days. Group II6 (test). Two additional Heidenhain pouch dogs were studied. One received 0.03 and the other 0.07 ml/kg of antigastrin antibody intravenously for eleven days. Daily basal gastrin determination and gastrin and acid responses to food were measured before and on the fifth and tenth days after injections were begun. On the eleventh day after the injection, all dogs were sacrificed and mucosal specimens from fundus, antrum, duodenum, and jejunum were obtained for analysis of their gastrin content. Group Ill (acute study)
Two dogs were prepared with Heidenhain and isolated antral pouches, drained to the exterior by metal cannulas. Continuity of the gastrointestinal tract was restored by gastroduodenostomy. The dogs were allowed to recover for three weeks. On the day of the experiment, after a 1 hour baseline period, a Foley catheter was introduced into the antral pouch, which was then ir-
lho Amerkan Jownal ot Surgery
Gastric Secretion and Serum Gastrin
rigated continuously with a 0.5 per cent solution of acetylcholine, pH 7, for 4 hours. After 1 hour of continuous a&al irrigation, both dogs received an injection of 0.03 ml/kg of antigastrin antibody intravenously. In a separate study, one of the dogs received 0.1,0.2 ana0.4 ml of antigastrin antibody intravenously at 1 hour intervals. Serum samples were obtained for gastrin measurement before and at regular intervals after initiation of antral irrigation. Gastric juice was collected (and acid output measured) for 30 minutes before and at 15 minute intervals during antral irrigation. Control experiments in which normal rabbit serum was used instead of antigastrin antibody were carried out in both dogs. Radioimmunoassay Technic. Serum levels of gastrin were measured by a specific gastrin radioimmunoassay [4,5]. The antigastrin rabbit serum used in the assay was capable of binding 25 per cent (10 pg) of lz51 human synthetic gastrin I at a final dilution of 1:100,000. The assay employs the second antibody technic described by Odell, Rayford, and Ross [6] to separate bound from free lz51 gastrin. Gastrin was radioiodinated in the assay by means of the technic of Greenwood,and Hunter 171. Human synthetic gastrin I (Imperial Chemical Industries, Ltd) was used as a reference standard. After antigastrin antibody injection, serum gastrin concentrations could not be estimated by our assay method in some dogs since the excess antigastrin antibody interfered with the assay (the serum samples bound labeled gastrin in the absence of added antigastrin serum). Serum gastrin in these samples were arbitrarily reported as undetectable. Bioassay. Gastric secretion from Heidenhain pouches was collected at 15 minute intervals and titrated against 0.1 N NaOH, using phenol red as an indicator. The results were expressed as mEq of HC1 for a 3 hour period. Results are expressed as the mean f one standard error. Student’s t test was used to analyze the data for statistical significance of differences between means. Differences with a p value of less than 0.05 were considered significant.
Results Group Ia. Basal gastrin levels before injection of antigastrin antibody ranged from 53 to 143 pg/ml, but gastrin was not measurable in our radioimmunoassay on the same day that antigastrin antibody was administered. However, basal gastrin was measurable on the next day (day 1) in the dog that received 0.001 ml/kg of antigastrin antibody; on day 4 in the dog that received 0.05 ml/kg; on day 2 in the one injected with 0.01 ml/kg; and not until day 9 in the dog that received the largest dose of 0.03 ml/kg of antigastrin antibody. (Figure 1.) Gastrin response to food and gastric acid output were normal before injection in all dogs. The day after injection of 0.001 ml/kg of antigastrin anti-
Volume 131, January 1976
MlN”TES
Figure 2. Serum gastrin levels and Het&nh8in pouch add wtput in one dog In responw to tkwd, before and afler a sh@s Injection of 0.001 ml/kg of ant&8Sttln antibody.
body, gastrin levels increased in response to food, but this value was slightly lesser than those gastrin concentrations measured before the injection; by day 3, gastrin values in response to food were greater than before injection. (Figure 2.) The dog injected with 0.03 ml/kg of antigastrin antibody had a normal gastrin response to food before injection of antigastrin antibody; after injection, the dog showed no gastrin response to food on day 1 or day 3; it was not until testing on day 10 that a normal response was seen. (Figure 3.) Heidenhain pouch acid output in response to food did not change significantly throughout the test periods in all the dogs. Group Ib. In both dogs without Heidenhain pouches basal gastrin levels were within normal limits before injection of antigastrin antibody. Basal gastrin could not be determined until day 26 in the dog that received 0.03 ml/kg of antigastrin antibody. In the dog that received 0.07 ml/kg, gastrin levels were first detected on day 47.
85
L
I
tevete and Hetdenhaln pouch actd cutput tn cne dog In response to feed, before and after in@ctton of 0.03 ml/kg of anttgaetrtn ant/body. F@we 3. Swum g&r/n
tents of the fundus, antrum, duodenum, and jejunum are shown in Table II, Group ZZZ. Mean basal gastrin level was 100 pg/ml and increased to a mean of 300 pg/ml after acetylcholine stimulation. The injection of 0.03 ml/kg of antigastrin antibody made gastrin undetectable one minute after its injection in both dogs. Basal acid secretory output was 0.3 mEq/hr and increased to 12.2 mEq/hr during stimulation with acetylcholine. Mean gastric acid output after injection of 0.03 ml/kg of antigastrin antibody was 14.1 mEq/hr. In the dog that received a bolus of 0.1, 0.2 and 0.4 ml/kg of antigastrin antibody on a separate day, gastric acid output decreased from 9.1 mEq/ hr during the period of stimulation alone to 6.1 mEq/hr after 0.1 ml/kg, 3.7 mEq/hr after 0.2 ml/ kg, and 2.17 mEq/hr after 0.4 ml/kg of antigastrin antibody. (Figure 5.1 Control studies in dogs receiving normal rabbit serum demonstrated a plateau of acid output. In the dog given antigastrin antibody, stimulation of the antral pouch with acetylcholine the next day yielded a normal gastric secretory response. Comments
F&we 4. Hektenhah pouch add output: per cent of the pretnjectbn vatue. lluee doge were gtven 0.03 ml/kg of rwMna/ rabbtt &num and one dcg was given 0.07 ml/kg; one dog was ghfen 0.03 mWkg of ant@etrln antibcdy and another 0.07 ml/kg.
Group ZZu.In the control group, daily basal gastrin level was unchanged during the eleven days, regardless of the amount of normal rabbit serum injected. Gastrin response to food was normal before and on days 5 and 10 after injection. The mean total amounts of acid released in response to food, before the injection and on days 5 and 10 after injection, are shown in Table I. The percentage of acid output of the preinjection value for the dogs injected with two dose levels of normal rabbit serum and two dose levels of antigastrin antibody is shown in Figure 4. The mean tissue gastrin contents of the fundus, antrum, duodenum, and jejunum are shown in Table II. Group ZZb. In the test group, basal gastrin levels were within normal limits before injection of antigastrin antibody. Immediately after injection neither basal nor food-stimulated gastrin levels could be measured in our assay. Gastric acid output was basically unchanged. (Table I.) Gastrin tissue con-
66
The association of peptic ulcer disease with gastric hypersecretion is firmly established. The classic treatment of peptic ulcer disease is aimed at reduction of acid output. Control of gastric acid secretion may be obtained with simple measures, such as neutralization with antacids, or may require removal of the end-organ (total gastrectomy in the case of patients with the Zollinger-Ellison syndrome). It has been proposed that there are three receptors in the parietal cell responsible for control of acid secretion: a receptor for acetylcholine, a receptor for histamine, and a receptor for gastrin [8]. Blockade of one of these receptors may change the properties of one or both of the other receptors and may diminish reactivity of the parieta1 cell. The idea of blocking or partially inactivating the action of the gastrin molecule is an attractive one, since gastrin plays a central role in the physiologic control of gastric secretion. Our results show that the administration of small doses of antigastrin antibody interferes with our ability to detect circulating gastrin in the radioimmunoassay system. This impairment may last as long as forty-seven days after a single intravenous injection of antigastrin antibody. Interference is clearly dose-related, depending on the balance between circulating gastrin and the amount TheAmerican
Journal ol Sutcntv
Gastric Secretionand SerumGastrin TABLE I
Total Heidenhain Pouch Acid Output in Response to Food in Group II (mEq/3 hours)
Injection Normal rabbit serum (control) 0.03 ml/kg (n = 3) 0.07 ml/kg (n = 1) Antigastrin antibody (test) 0.03 ml/kg (n = 1) 0.07 ml/kg (n = 1)
Preinjection
6.53 3.40
11.3 1.8
Day 5
Day 10
7.4 3.5
9.4 2.9
11.4 1.0
8.1 1.2
of antibody present. Basal levels of gastrin could not be detected after administration of antigastrin antibody in the first group of dogs, but gastrin in response to food stimulation was measured the same day. These findings presumably are due to interference caused by the circulating antibody, which binds the labeled gastrin in the assay. Control values for mucosal concentration of gastrin are in agreement with previously reported values [9,10]. Mucosal content of gastrin was much greater in dogs injected with antigastrin antibody, even though circulating gastrin could not be detected. It is possible that the circulating antigastrin antibody acted in some way to stimulate synthesis or to interfere with the release of gastrin. These are not likely to be the only reasons, since tissue concentrations of gastrin were also increased in fundic mucosa. Another possible explanation may be that during the process of gastrin tissue extraction (which includes boiling of the tissue for 30 minutes), the antigastrin antibody complex was destroyed, so that gastrin bound to antibody was liberated and available for measurement. In a preliminary, unpublished study, performed in conjunction with Doctor Juan Lechago, no ultrastructural changes in G cells have been detected in specimens taken from dogs in group IIb. Jaffe, Newton, and McGuigan demonstrated that preincubation of synthetic human gastrin I and pentagastrin with antigastrin antibody, inhibited gastric secretory activity in rate. Furthermore, direct administration of antigastrin antibody to rate inhibited the acid secretory effects of endogenously released gastrin [II]. Gastric acid output was diminished in a single patient with the Zollinger-Ellison syndrome who received one dose of antigastrin antibody (of a potency similar to that used in the present study) [I]. Our results show that a massive injection of antigastrin antibody was required to produce significant gastric acid inhibition and even then, the effect was transitory, lasting less than 24 hours.
Vokmw 131, Jamiuy lD76
TABLE II
Tissue Gastrin Content in Group II (ng/g)
Normal rabbit serum (control) 0.03 ml/kg (n = 3) 0.07 ml/kg (n = 1) Antigastrin antibody (test) 0.03 ml/kg (n = 1) 0.07 ml/kg (n = 1)
30
Antrum
denum
Jejunum
15.2 56
9,200 16,400
196 184
132 190
25,000 28.700
302 378
246 144
102 176
IRRIGATION
O
Fundus
DUO-
Injection
WITU
90
ACETYLCHDLINE
I20
Is0
-I
lw
2D
240
MINUTES
Figun, 5. EtYact of vmyhg doees of nomad raM# and ant-k, antbody on acdyMotb&~ted trhz a&t secret&n.
serum gas-
Therapeutic use of antigastrin antibody to control gastric acid secretion does not seem feasible to us at the present time because of the scarcity of the antigastrin antibody and because large doses are required to obtain only a temporary antisecretory effect.
Fourteen dogs received varying doses of antigastrin antibody; after a single dose of 0.07 ml/kg, circulating gastrin levels could not be measured for as long as forty-seven days. Gastric secretion in response to food was not diminished by antigastrin antibody doses as high as 0.07 ml/kg daily for ten days. Larger doses, 0.2 and 0.4 ml/kg, were required to produce a temporary reduction in gastric-stimulated gastric acid secretion. Mucosal levels of gastrin in the antrum, fundus, and duodenum were greatly increased ten days after injection of antigastrin antibody. The therapeutic use of antigastrin antibody to control gastric secretion seems at the present time not feasible because of the scarcity of the antigastrin antibody and because large doses are required to obtain only a temporary effect. 67
Vilbr et al
References 1. Hughes WS. Hughes JB: Use of gastrin antibody in the ZolIinger-Ellison syndrome. C/in Res 21: 51. 1973. 2. Jaffe BM, Newton WT. M&&an JM: Inhibition of gastrin activity by incubation with antibodies to the C-terminal tetrapeptii of gasbin. Surgery 85: 633, 1969. 3. Rayford PL, Vaitukattis JL. Ross GT, Morgan FJ, Canfield RE: Use of specific antisera to characterize biologic activity of hCH fi subunit preparations. fndocrinofogy91: 144, 1972. 4. Jackson BM, Reeder DD. Thompson JC: Dynamic characteristics of gastrin release. Am J Surg 123: 137, 1972. 5. Aayford PL. Reeder. DD, Thompson JC: lnteriaboratory reproducibility of gastrin measurements by radioimmunoassay. J Lab C/in Msd in press. 6. Odall WD, Rayford PL. Ross GT: Simplified. partially automated method for radioimmunoassay of human thyroid stimulating growth, luteiniring and follicle stimulating hormones. J Lab C/in Med 70: 973, 1967. 7. tieenwood FC, Hunter WM: The preparation of ‘311-labeled human growth hormone of hi specific radioactivity. Biothem J89: 114, 1963. 6. Grossman Ml, Konturek SJ: Inhibition of acid secretion in dog by methmkb. a histamine antagonist acting on Hz receptors. Gastrmnterology66: 517. 1974. 9. Watson LC. Reeder DD. Becker HD, LaGrone L, Thompson JC: Gastrin concentration in upper gastrointestinal mucosa in dogs. Surgery76: 419, 1974. 10. Jackson BM, ReeUer DD, Searcy JR, Watson LC, Hirose FM, Thompson JC: Correlation of the surface pli. histology arid gastrin concentration of gastric mucosa. Ann Surg 176: 727, 1972. 11. Jaffe BM, Newton Wl, McGuigan JE: Inhibition of endogeM)uS gastrin activii by antibodies lo the carboxyl-terminal tetrapeptide amide of gastrin. Gastrcentefobgy 58: 151,
1970.
Discussion Chairman William Silen (Boston, MA): I have a question, Doctor Villar. If you add exogenous gastrin
when you have this level, which is undetectable, at the time when the stomach is still responding, if you gave that animal exogenous gastrin, can you measure this in the blood? In the plasma? Hugo V. Villar: Chairman
68
I doubt it very much.
Silen:
Do we have what the threshold
level for secretion is as far as gastrin is concerned? In other words, what level in the bloodstream does one need to see secretion? James C, Thompson: It is a question of change, not absolute level, and a change of 5 pg will do it. Robert Zeppa (Miami, FA): In most animals, we were able with massive doses to block acid secretion. Were any of these animals challenged with histamine? Doctor Villar: Yes, all were tested before and after and there was no change. Doctor Zeppa: Even at the time pressed for other reasons?
they were sup-
Doctor Villar: Not at the same time but the next day, as in the dog in which we did suppress the acid secretions, then the output was completely normal; so there is no damage. Chairman Silen: To put this in some perspective, if we are dealing with this animal who has been immunized against gastrin, isn’t there a marvelous preparation to look at other aspects of gastric secretions? Doctor Villar: It surely would be immunized against gastrin to protect some patients, such as patients with Zollinger-Ellison syndrome patients; this is certainty attractive and Doctor Jaffe did similar studies in the rat about four or five years ago, when he did show inhibition. We were unable to find any significant inhibition at the doses tested, but the possibility is there. Josef E. Fischer (Boston, MA): The fact you were unable to detect gastrin in the circulation may be artifactual, because of an excess of circulating antibody. l do not know what its half-life is-possibly greater than ten days-so that we may have circulating gastrin and it may be bound and it may be equilibrium and it may be able to influence gastric secretion. Doctor Villar (closing): We gave two dogs a single shot and we could not detect gastrin in one until day 47. SO it has a half-life as long as forty-seven days.
The Am&can Journalot Surgery
Robert A. D. Booth, FRCS, Galveston, Texas Phillip L. Rayford, PhD, Galveston, Texas David D. Reeder, MD, Galveston, Texas James C. Thompson, MD, Galveston, Texas
Neutralization of circulating gastrin, which would result in diminution in gastric acid secretion, might be effective in the treatment of peptic ulcer disease, especially in patients with the ZollingerEllison syndrome. A preliminary report [I] suggested that gastrin could be neutralized by the administration of antigastrin antibody (Abe) in man. Incubation of gastrin with specific antibody in vitro, followed by administration of the mixture to rats, has been shown to abolish biologic activity of gastrin (21. Inactivation of hormone by antibody in vitro is related to the dose of hormone and the concentration of antibody used [3]. In order to learn more about the mechanism of hormonal inactivation in vivo, we have studied the effects on gastric secretion and on circulating serum gastrin (as measured by radioimmunoassay) of varying doses of either normal rabbit serum or antigastrin antibody administered to dogs. Material and Methods Production of Antigastrin Antibody. Synthetic human gastrin (2 to 17 amino acids) was used as an immunogen to generate antigastrin antibody in New Zealand white rabbits. The rabbits were bled weekly, and serum was collected and tested for antibody titer. A pool of undiluted antigastrin antibody (found to bind approximately 1.7 pg synthetic human gastrin per ml by radioimmunoassay) was used for these studies. Fourteen healthy mongrel dogs were divided into three experimental groups. Group I (chronic study, single Injection)
Group la. Four dogs were prepared pouches. Three weeks after recovery,
with Heidenhain each dog received
From the Department of Surgery, The University of Texas Medical Branch. Wveston. Texas. This work was suppcfted by a grant from the National Institutes of Health (AM 15241). Reprint requests should be addressed to James C Thompson, MD, Department of Surgery. The University of Texas Medical Branch. Galveston, Texas 77550. Presented at the Sixteenth Annual Meeting of the Society for Surgery of the Alimentary Tract, San Antonio, Texas, May 20-21. 1975.
64
a single dose of either 0.001, 0.005, 0.01 or 0.03 ml/kg of Abe intravenously. Basal blood samples for serum gastrin determination were drawn daily one week before and for two weeks after injection of the antigastrin antibody. Two food studies were performed before the day of injection, and one, three, and ten days after the injection of antigastrin antibody. At each study, blood samples for gastrin were drawn before feeding a standard 400 gm meat meal and at 5, 10, 15, 30, 60, 90, 120, and 180 minutes after feeding. Gastric secretions were collected at 15 minute intervals for 180 minutes throughout the test period. Group lb. Two additional dogs without Heidenhain pouches were injected with a single dose of 0.03 and 0.07 ml/kg of antigastrin antibody. Basal blood samples for gastrin determinations were drawn before and daily after injection for four months. Group [I (chronk study, multiple Injection)
Group Ila (control). Four dogs were prepared with Heidenhain pouches. Three weeks after recovery, three of them received a daily injection of 0.03 ml/kg of normal rabbit serum for eleven days intravenously. The fourth dog received 0.07 ml/kg of normal rabbit serum for eleven days. Group II6 (test). Two additional Heidenhain pouch dogs were studied. One received 0.03 and the other 0.07 ml/kg of antigastrin antibody intravenously for eleven days. Daily basal gastrin determination and gastrin and acid responses to food were measured before and on the fifth and tenth days after injections were begun. On the eleventh day after the injection, all dogs were sacrificed and mucosal specimens from fundus, antrum, duodenum, and jejunum were obtained for analysis of their gastrin content. Group Ill (acute study)
Two dogs were prepared with Heidenhain and isolated antral pouches, drained to the exterior by metal cannulas. Continuity of the gastrointestinal tract was restored by gastroduodenostomy. The dogs were allowed to recover for three weeks. On the day of the experiment, after a 1 hour baseline period, a Foley catheter was introduced into the antral pouch, which was then ir-
lho Amerkan Jownal ot Surgery
Gastric Secretion and Serum Gastrin
rigated continuously with a 0.5 per cent solution of acetylcholine, pH 7, for 4 hours. After 1 hour of continuous a&al irrigation, both dogs received an injection of 0.03 ml/kg of antigastrin antibody intravenously. In a separate study, one of the dogs received 0.1,0.2 ana0.4 ml of antigastrin antibody intravenously at 1 hour intervals. Serum samples were obtained for gastrin measurement before and at regular intervals after initiation of antral irrigation. Gastric juice was collected (and acid output measured) for 30 minutes before and at 15 minute intervals during antral irrigation. Control experiments in which normal rabbit serum was used instead of antigastrin antibody were carried out in both dogs. Radioimmunoassay Technic. Serum levels of gastrin were measured by a specific gastrin radioimmunoassay [4,5]. The antigastrin rabbit serum used in the assay was capable of binding 25 per cent (10 pg) of lz51 human synthetic gastrin I at a final dilution of 1:100,000. The assay employs the second antibody technic described by Odell, Rayford, and Ross [6] to separate bound from free lz51 gastrin. Gastrin was radioiodinated in the assay by means of the technic of Greenwood,and Hunter 171. Human synthetic gastrin I (Imperial Chemical Industries, Ltd) was used as a reference standard. After antigastrin antibody injection, serum gastrin concentrations could not be estimated by our assay method in some dogs since the excess antigastrin antibody interfered with the assay (the serum samples bound labeled gastrin in the absence of added antigastrin serum). Serum gastrin in these samples were arbitrarily reported as undetectable. Bioassay. Gastric secretion from Heidenhain pouches was collected at 15 minute intervals and titrated against 0.1 N NaOH, using phenol red as an indicator. The results were expressed as mEq of HC1 for a 3 hour period. Results are expressed as the mean f one standard error. Student’s t test was used to analyze the data for statistical significance of differences between means. Differences with a p value of less than 0.05 were considered significant.
Results Group Ia. Basal gastrin levels before injection of antigastrin antibody ranged from 53 to 143 pg/ml, but gastrin was not measurable in our radioimmunoassay on the same day that antigastrin antibody was administered. However, basal gastrin was measurable on the next day (day 1) in the dog that received 0.001 ml/kg of antigastrin antibody; on day 4 in the dog that received 0.05 ml/kg; on day 2 in the one injected with 0.01 ml/kg; and not until day 9 in the dog that received the largest dose of 0.03 ml/kg of antigastrin antibody. (Figure 1.) Gastrin response to food and gastric acid output were normal before injection in all dogs. The day after injection of 0.001 ml/kg of antigastrin anti-
Volume 131, January 1976
MlN”TES
Figure 2. Serum gastrin levels and Het&nh8in pouch add wtput in one dog In responw to tkwd, before and afler a sh@s Injection of 0.001 ml/kg of ant&8Sttln antibody.
body, gastrin levels increased in response to food, but this value was slightly lesser than those gastrin concentrations measured before the injection; by day 3, gastrin values in response to food were greater than before injection. (Figure 2.) The dog injected with 0.03 ml/kg of antigastrin antibody had a normal gastrin response to food before injection of antigastrin antibody; after injection, the dog showed no gastrin response to food on day 1 or day 3; it was not until testing on day 10 that a normal response was seen. (Figure 3.) Heidenhain pouch acid output in response to food did not change significantly throughout the test periods in all the dogs. Group Ib. In both dogs without Heidenhain pouches basal gastrin levels were within normal limits before injection of antigastrin antibody. Basal gastrin could not be determined until day 26 in the dog that received 0.03 ml/kg of antigastrin antibody. In the dog that received 0.07 ml/kg, gastrin levels were first detected on day 47.
85
L
I
tevete and Hetdenhaln pouch actd cutput tn cne dog In response to feed, before and after in@ctton of 0.03 ml/kg of anttgaetrtn ant/body. F@we 3. Swum g&r/n
tents of the fundus, antrum, duodenum, and jejunum are shown in Table II, Group ZZZ. Mean basal gastrin level was 100 pg/ml and increased to a mean of 300 pg/ml after acetylcholine stimulation. The injection of 0.03 ml/kg of antigastrin antibody made gastrin undetectable one minute after its injection in both dogs. Basal acid secretory output was 0.3 mEq/hr and increased to 12.2 mEq/hr during stimulation with acetylcholine. Mean gastric acid output after injection of 0.03 ml/kg of antigastrin antibody was 14.1 mEq/hr. In the dog that received a bolus of 0.1, 0.2 and 0.4 ml/kg of antigastrin antibody on a separate day, gastric acid output decreased from 9.1 mEq/ hr during the period of stimulation alone to 6.1 mEq/hr after 0.1 ml/kg, 3.7 mEq/hr after 0.2 ml/ kg, and 2.17 mEq/hr after 0.4 ml/kg of antigastrin antibody. (Figure 5.1 Control studies in dogs receiving normal rabbit serum demonstrated a plateau of acid output. In the dog given antigastrin antibody, stimulation of the antral pouch with acetylcholine the next day yielded a normal gastric secretory response. Comments
F&we 4. Hektenhah pouch add output: per cent of the pretnjectbn vatue. lluee doge were gtven 0.03 ml/kg of rwMna/ rabbtt &num and one dcg was given 0.07 ml/kg; one dog was ghfen 0.03 mWkg of ant@etrln antibcdy and another 0.07 ml/kg.
Group ZZu.In the control group, daily basal gastrin level was unchanged during the eleven days, regardless of the amount of normal rabbit serum injected. Gastrin response to food was normal before and on days 5 and 10 after injection. The mean total amounts of acid released in response to food, before the injection and on days 5 and 10 after injection, are shown in Table I. The percentage of acid output of the preinjection value for the dogs injected with two dose levels of normal rabbit serum and two dose levels of antigastrin antibody is shown in Figure 4. The mean tissue gastrin contents of the fundus, antrum, duodenum, and jejunum are shown in Table II. Group ZZb. In the test group, basal gastrin levels were within normal limits before injection of antigastrin antibody. Immediately after injection neither basal nor food-stimulated gastrin levels could be measured in our assay. Gastric acid output was basically unchanged. (Table I.) Gastrin tissue con-
66
The association of peptic ulcer disease with gastric hypersecretion is firmly established. The classic treatment of peptic ulcer disease is aimed at reduction of acid output. Control of gastric acid secretion may be obtained with simple measures, such as neutralization with antacids, or may require removal of the end-organ (total gastrectomy in the case of patients with the Zollinger-Ellison syndrome). It has been proposed that there are three receptors in the parietal cell responsible for control of acid secretion: a receptor for acetylcholine, a receptor for histamine, and a receptor for gastrin [8]. Blockade of one of these receptors may change the properties of one or both of the other receptors and may diminish reactivity of the parieta1 cell. The idea of blocking or partially inactivating the action of the gastrin molecule is an attractive one, since gastrin plays a central role in the physiologic control of gastric secretion. Our results show that the administration of small doses of antigastrin antibody interferes with our ability to detect circulating gastrin in the radioimmunoassay system. This impairment may last as long as forty-seven days after a single intravenous injection of antigastrin antibody. Interference is clearly dose-related, depending on the balance between circulating gastrin and the amount TheAmerican
Journal ol Sutcntv
Gastric Secretionand SerumGastrin TABLE I
Total Heidenhain Pouch Acid Output in Response to Food in Group II (mEq/3 hours)
Injection Normal rabbit serum (control) 0.03 ml/kg (n = 3) 0.07 ml/kg (n = 1) Antigastrin antibody (test) 0.03 ml/kg (n = 1) 0.07 ml/kg (n = 1)
Preinjection
6.53 3.40
11.3 1.8
Day 5
Day 10
7.4 3.5
9.4 2.9
11.4 1.0
8.1 1.2
of antibody present. Basal levels of gastrin could not be detected after administration of antigastrin antibody in the first group of dogs, but gastrin in response to food stimulation was measured the same day. These findings presumably are due to interference caused by the circulating antibody, which binds the labeled gastrin in the assay. Control values for mucosal concentration of gastrin are in agreement with previously reported values [9,10]. Mucosal content of gastrin was much greater in dogs injected with antigastrin antibody, even though circulating gastrin could not be detected. It is possible that the circulating antigastrin antibody acted in some way to stimulate synthesis or to interfere with the release of gastrin. These are not likely to be the only reasons, since tissue concentrations of gastrin were also increased in fundic mucosa. Another possible explanation may be that during the process of gastrin tissue extraction (which includes boiling of the tissue for 30 minutes), the antigastrin antibody complex was destroyed, so that gastrin bound to antibody was liberated and available for measurement. In a preliminary, unpublished study, performed in conjunction with Doctor Juan Lechago, no ultrastructural changes in G cells have been detected in specimens taken from dogs in group IIb. Jaffe, Newton, and McGuigan demonstrated that preincubation of synthetic human gastrin I and pentagastrin with antigastrin antibody, inhibited gastric secretory activity in rate. Furthermore, direct administration of antigastrin antibody to rate inhibited the acid secretory effects of endogenously released gastrin [II]. Gastric acid output was diminished in a single patient with the Zollinger-Ellison syndrome who received one dose of antigastrin antibody (of a potency similar to that used in the present study) [I]. Our results show that a massive injection of antigastrin antibody was required to produce significant gastric acid inhibition and even then, the effect was transitory, lasting less than 24 hours.
Vokmw 131, Jamiuy lD76
TABLE II
Tissue Gastrin Content in Group II (ng/g)
Normal rabbit serum (control) 0.03 ml/kg (n = 3) 0.07 ml/kg (n = 1) Antigastrin antibody (test) 0.03 ml/kg (n = 1) 0.07 ml/kg (n = 1)
30
Antrum
denum
Jejunum
15.2 56
9,200 16,400
196 184
132 190
25,000 28.700
302 378
246 144
102 176
IRRIGATION
O
Fundus
DUO-
Injection
WITU
90
ACETYLCHDLINE
I20
Is0
-I
lw
2D
240
MINUTES
Figun, 5. EtYact of vmyhg doees of nomad raM# and ant-k, antbody on acdyMotb&~ted trhz a&t secret&n.
serum gas-
Therapeutic use of antigastrin antibody to control gastric acid secretion does not seem feasible to us at the present time because of the scarcity of the antigastrin antibody and because large doses are required to obtain only a temporary antisecretory effect.
Fourteen dogs received varying doses of antigastrin antibody; after a single dose of 0.07 ml/kg, circulating gastrin levels could not be measured for as long as forty-seven days. Gastric secretion in response to food was not diminished by antigastrin antibody doses as high as 0.07 ml/kg daily for ten days. Larger doses, 0.2 and 0.4 ml/kg, were required to produce a temporary reduction in gastric-stimulated gastric acid secretion. Mucosal levels of gastrin in the antrum, fundus, and duodenum were greatly increased ten days after injection of antigastrin antibody. The therapeutic use of antigastrin antibody to control gastric secretion seems at the present time not feasible because of the scarcity of the antigastrin antibody and because large doses are required to obtain only a temporary effect. 67
Vilbr et al
References 1. Hughes WS. Hughes JB: Use of gastrin antibody in the ZolIinger-Ellison syndrome. C/in Res 21: 51. 1973. 2. Jaffe BM, Newton WT. M&&an JM: Inhibition of gastrin activity by incubation with antibodies to the C-terminal tetrapeptii of gasbin. Surgery 85: 633, 1969. 3. Rayford PL, Vaitukattis JL. Ross GT, Morgan FJ, Canfield RE: Use of specific antisera to characterize biologic activity of hCH fi subunit preparations. fndocrinofogy91: 144, 1972. 4. Jackson BM, Reeder DD. Thompson JC: Dynamic characteristics of gastrin release. Am J Surg 123: 137, 1972. 5. Aayford PL. Reeder. DD, Thompson JC: lnteriaboratory reproducibility of gastrin measurements by radioimmunoassay. J Lab C/in Msd in press. 6. Odall WD, Rayford PL. Ross GT: Simplified. partially automated method for radioimmunoassay of human thyroid stimulating growth, luteiniring and follicle stimulating hormones. J Lab C/in Med 70: 973, 1967. 7. tieenwood FC, Hunter WM: The preparation of ‘311-labeled human growth hormone of hi specific radioactivity. Biothem J89: 114, 1963. 6. Grossman Ml, Konturek SJ: Inhibition of acid secretion in dog by methmkb. a histamine antagonist acting on Hz receptors. Gastrmnterology66: 517. 1974. 9. Watson LC. Reeder DD. Becker HD, LaGrone L, Thompson JC: Gastrin concentration in upper gastrointestinal mucosa in dogs. Surgery76: 419, 1974. 10. Jackson BM, ReeUer DD, Searcy JR, Watson LC, Hirose FM, Thompson JC: Correlation of the surface pli. histology arid gastrin concentration of gastric mucosa. Ann Surg 176: 727, 1972. 11. Jaffe BM, Newton Wl, McGuigan JE: Inhibition of endogeM)uS gastrin activii by antibodies lo the carboxyl-terminal tetrapeptide amide of gastrin. Gastrcentefobgy 58: 151,
1970.
Discussion Chairman William Silen (Boston, MA): I have a question, Doctor Villar. If you add exogenous gastrin
when you have this level, which is undetectable, at the time when the stomach is still responding, if you gave that animal exogenous gastrin, can you measure this in the blood? In the plasma? Hugo V. Villar: Chairman
68
I doubt it very much.
Silen:
Do we have what the threshold
level for secretion is as far as gastrin is concerned? In other words, what level in the bloodstream does one need to see secretion? James C, Thompson: It is a question of change, not absolute level, and a change of 5 pg will do it. Robert Zeppa (Miami, FA): In most animals, we were able with massive doses to block acid secretion. Were any of these animals challenged with histamine? Doctor Villar: Yes, all were tested before and after and there was no change. Doctor Zeppa: Even at the time pressed for other reasons?
they were sup-
Doctor Villar: Not at the same time but the next day, as in the dog in which we did suppress the acid secretions, then the output was completely normal; so there is no damage. Chairman Silen: To put this in some perspective, if we are dealing with this animal who has been immunized against gastrin, isn’t there a marvelous preparation to look at other aspects of gastric secretions? Doctor Villar: It surely would be immunized against gastrin to protect some patients, such as patients with Zollinger-Ellison syndrome patients; this is certainty attractive and Doctor Jaffe did similar studies in the rat about four or five years ago, when he did show inhibition. We were unable to find any significant inhibition at the doses tested, but the possibility is there. Josef E. Fischer (Boston, MA): The fact you were unable to detect gastrin in the circulation may be artifactual, because of an excess of circulating antibody. l do not know what its half-life is-possibly greater than ten days-so that we may have circulating gastrin and it may be bound and it may be equilibrium and it may be able to influence gastric secretion. Doctor Villar (closing): We gave two dogs a single shot and we could not detect gastrin in one until day 47. SO it has a half-life as long as forty-seven days.
The Am&can Journalot Surgery
