GASTROENTEROLOGY Copyright 1969 by The Williams & Wilkins Co. Vol. 56, No.3 Printea in U.S.A. MECHANISM BY WHICH FAT IN THE UPPER SMALL INTESTINE INHIBITS GASTRIC ACID H. T. DEBAS, M.D., B. S. BEDI, M.B., F.R.C.S., G. GILLESPIE, M.B., F.R.C.S., AND I. E. GILLESPIE, M.D., F.R.C.S. University Department 01 Surgery, Western Infirmary, Glasgow, Scotland The inhibition caused by fat in contact with the duodenum and upper small intestine appears to be equally effective against endogenous and exogenous gastrin. Therefore, it is suggested that the humoral agent released by fat acts predominantly, or solely by inactivating circulating gastrin, rather than by preventing gastrin release from the pyloric gland area. Various substances, including fats, coming in contact with the mucosa of the duodenum and upper jejunum exert an inhibitory influence on gastric acid responses. The demonstration that duodenal irrigation by fat suppressed the acid secretion of transplanted gastric pouches (Feng et al. I) indicated that the inhibition was humorally mediated and Kosaka and Lim 2 proposed the name "enterogastrone" for the humoral inhibitor agent. Gregory and Tracy3 have suggested that the inhibition from fat in the duodenum acts by preventing the release of gastrin from the pyloric gland area. Gillespie and Grossman 4 found that feeding a fat emulsion inhibited the acid' responses of pouch dogs to the continuous intravenous infusion of relatively crude gastrin extracts. The principal aim of the present study was to compare in the same animals the effectiveness of fat in tne upper small intestine against both endogenously released gastrin, and both pure Received July 8, 1968. Accepted September 11, 1968. Address requests for reprints to: Dr. I. E. Gilles pie, University Department of Surgery, Western Infirmary, Glasgow, W.1, Scotland. The authors are very grateful to Dr. J. D. Fitzgerald, of Imperial Chemical Industries, Limited, for supplying the pentagastrin, and Professor R. A. Gregory, of Liverpool University, for supplying the pure gastrin II extract used in these experiments. 483 gastrin and the synthetic gastrin-like pentapeptide, pentagastrin, by injection. The acid responses to both endogenous and exogenous gastrin were matched so that the effects of the fat would be assessed against equivalent degrees of gastrin stimulation. A comparable degree of stimulation by infused histamine was also used in a final group of experiments in the same animals. Methods and Materials Three mongrel dogs, weighing between 15 and 20 kg, and each with a vagally innervated pouch of the pyloric gland area and a Heidenhain-type denervated fundic pouch, were used. The pyloric gland area pouch was separated from the proximal stomach by a double mucosal septum, constructed through a short transverse incision on the greater curvature. The transected pylorus was brought out to the surface of the abdominal wall as a mucocutaneous fistula. Gastrointestinal continuity was restored by an anastomosis between the stomach just proximal to the double mucosal septum, and the first part of the duodenum. No secretory observations were made until at least 1 month had elapsed after the operative procedures. The dogs remained at constant weight and in good health throughout the series of experiments. Intact vagal innervation of the pyloric gland area pouch was verified in each dog by the demonstration of an acid response from the Heidenhain pouch after the intravenous injection of units of soluble insulin.
484 DEBAS ET AL. Vol. 56, No.3 The "maximal" histamine response of each Heidenhain pouch was first determined by doubling the dose of a continuous intravenous infusion of histamine acid phosphate until no further increase in acid output was obtained. Irrigation of the pyloric gland area pouch with acetylcholine chloride solution was used to cause the endogenous release of gastrin. The solution was introduced into the pouch at a constant rate of 42 ml per hr by means of a Palmer infusion pump. In preliminary control runs, a concentration of acetylcholine chloride was selected to give Heidenhain pouch acid responses between 40 and 50% of the maximal histamine response. In 2 dogs, 0.1 Co acetylcholine chloride solution gave this level of acid output, and in the 3rd 0.550 solution was required. The ph of the irrigating solution was always adjusted to be not less than 6.5. Exogenous gastrin stimulation was provided in most experiments by the continuous intravenous infusion of pentagastrin in doses ranging from to 15 Jig per hr, and in other experiments of Jig per hr pure gastrin II extract prepared by the technique of Gregory and Tracy." In the histamine experiments the acid phosphate, in a dose selected to give acid responses comparable to both gastrin series, ranging from 0.12 to 0.76 mg per hr, was given by continuous intravenous infusion. Both gastrin preparations and histamine were made up in appropriate concentration with 0.9S;: sodium chloride solution, and given by continous constant-rate intravenous infusion by means of a Palmer pump, at a rate of 21 mlper hr. At least three experimental runs were performed with each stimulant on each dog. Occasional signs suggestive of nausea or retching occurred at the onset of irrigation of the pyloric gland area pouch, and these experiments were abandoned. All of those in which no such signs appeared at any time on continuous close observation were included in the analyses. Acid secretion from the Heidenhain pouch was collected continuously in each experiment, and every 15 min the volume was measured in milliliters and the acid concentration estimated by titration against 0.01 N sodium hydroxide, with phenol red indicator. Acid output was derived from multiplication of the volume and concentration values. All outputs were calculated as milliequivalents per 15- min period, and expressed in figures 1 to 4 as percentages, taking control plateau levels as 0%. A plateau of acid secretion from the Heidenhain pouch either to irrigation of the pyloric gland area pouch or to intravenous infusion of stimulant was considered to have been achieved when three consecutive 15-min acid outputs varied by less than 15%. Such a plateau was obtained in most experiments by 75 min, and in all by 120 min. The fat used was a commercially available corn oil (Mazola), which contained 59% polyunsaturated fatty acids. Mter the plateau Heidenhain pouch response had been established, three separate quantities of 20 ml of the oil were fed to the dogs every 5 min over a 15-min period. The animals lapped up the oil with relish and were seen to consume the entire volume offered on each occasion. Fifteen-minute collections of Heidenhain pouch responses were continued for 3 hr after administration of the oil. The statistical significance of the difference between each mean 15-min acid output after the oil, and the mean control plateau response, was calculated by Student's t-test for paired values. Results Matching of Heidenhain Pouch Acid For each individual dog the responses to pyloric gland area stimulation and to infused pentagastrin and histamine were comparable, although there was some variation in the output levels from dog to dog, as shown in table 1. TABLE 1. Acid responses of Heidenhain pouches to gastrin and histamine a Gastrin Histamine Dog Endogenous, Exogenous, (intravenous pyloric pouch, pentagastrin infusion) irrigation with (intravenous acetylcholine infusion) p.eq!15 min p.eq! 15 min 1 2 250 300 2 6 94 136 3 425 450 482 a Means of three to five experiments with each agent.
March 1969 FAT IN THE UPPER SMALL INTESTINE 485 Effect of Fat on Endogenous Gastrin The results of 13 experiments in the 3 dogs are shown in figure 1. Some degree of inhibition of the Heidenhain pouch response to acetylcholine irrigation of the pyloric gland area was apparent in the second 15-min period after feeding the corn oil and was statistically highly significant in the third 15-min period. Maximal inhibition, between 75 and 80% of the control plateau levels, occurred between 45 and min; thereafter, there was a tendency for the acid responses to return toward control levels. However, even 3 hr after feeding the corn oil, the inhibition of acid response Acid Output f'zl 0.% of Control + + 1 Lr_ loric Gland Area Pouch Control,.---,.. jiiii;;e~- "-- L... ; - l... r - ~-- L_.; t"' j was still significant. The reduction in acid output was reflected in reductions in both the volume of secretion and acid concentration. Effect of Fat on Exogenous Gastrin-like Preparation The means of 15 experiments using pentagastrin in the 3 dogs are shown In figure 2. Highly significant inhibition of the Heidenhain pouch acid response to the continuous intravenous infusion of pentagastrin occurred in the second 15- min period after feeding the corn oil. The degree and duration of inhibition were very similar to that observed in the endogenous gastrin release experiments. Maximal inhibition was again between and 80% of the control plateau levels, and significant inhibition lasted throughout a 3-hr period. The supply of pure gastrin II was relatively limited, and only one experimental run using this preparation was made in 50 30 o 13 Expts. 2 6 8 12 14 1..---, 1 50 n Control ""L.."'l,.. J'..' FIG. 1. Effect of 60 ml of com oil given orally over 15 min on Heidenhain p ouch acid response to irrigation of vagally innervated pyloric gland area pouch with acetylcholine (0.1 or 0. 5%). Control data from four experiments on the 3 dogs; 0% = 299 I'Eq per 15 min. In this figure, and figures 2, 3 and 4, the solid lines represent the means, and the shaded areas the standard errors of the means, o f all the experiments in this group. Asterisks denote statistically significant differences from control plateau values (* = P < 0.05, ** = P < 0.01 ; Stu dent's t test for paired values). Significant inhibi tion was shown from 30 min after oil ingestion to end of experiment, 3 hr later. 30 15 Expts. 2 4 6 8 12 14 FIG. 2. Effect of 60 ml of com oil given orally the continuous constant rate intravenous infusion of pentagastrin (doses selected to match outputs to pyloric gland area pouch irrigation experiments of fig. 1). Control data from one experiment on each of 3 dogs; 0% = 2651'Eq per 15 min.
486 DEBAS ETAL. Vol. 56, No.3 Acid Output ~ as % of Control 1 so 30.. Expts 2 Dogs t t 2 4 6 8 12 14 FIG. 3. Effect of 60 ml of com oil given orally the continuous constant-rate intravenous infusion of gastrin II (doses selected to match outputs in figures 1 and 2); 0 \'0 = 3681lEq per 15 min. ACid Output 0'4 of Cantrol SO 30 o 2 14 Expls. 15 min. Intenool. FIG. 4. Effect of 60 ml of com oil given orally the continuous constant-rate intravenous infusion of histamine acid phosphate (doses selected to match outputs in figures I, 2, and 3. Control data from one experiment on each of the 3 dogs; 0S;' = 293 IlEq per 15 min. each dog, the results of which are shown in figure 3. Our aim was simply to determine whether the effect of fat feeding on the response to infused pentagastrin was representative of the effect on what is probably the "physiological" circulating gastrin. A fixed dose of Ilg per hr of the pure gastrin extract was selected and, although there was a greater degree of individual variation in acid response from dog to dog, maximal inhibition was of the same order and the same duration as in the previous two groups of experiments. There was an insufficient supply of the gastrin II to perform control runs with this material alone on this occasion, but it was felt unnecessary to obtain a fresh supply for this purpose, since in previous experiments on these same dogs sustained plateau responses to the continuous intravenous infusion of pure gastrin II had been obtained regularly. Effect of Fat on Histamine-stimulated Means of 14 experiments in the 3 dogs are illustrated in figure 4. Although there was a slight tendency for the acid output to diminish below control values in the 2nd and 3rd hr after feeding the corn oil, at no time was there statistically significant inhibition. On the other hand, there was some evidence of slight augmentation of the acid response to histamine, which reached borderline significance in the second and third 15-min period after the corn oil. This effect was more constant and more marked in 1 of the 3 dogs than in the other 2. Discussion Fat coming in contact with the gastric mucosa has been shown to be without effect on gastric acid secretory responses by Shay and co-workers. 5 We are thus able to study the influence of fat in the upper small intestine simply by feeding the material and allowing it to come naturally into contact with the intestinal mucosa. The present experiments appear to confirm the finding of significant inhibition of gastric pouch acid responses to the intravenous infusion of pure gastrin
March 1969 FAT IN THE UPPER SMALL INTESTINE 487 extract, and pure synthetic gastrin-like pentapeptide. These results are likewise in keeping with a humoral mechanism of inhibition, since the effects were observed in denervated pouches separated from the main stomach. The inhibitory effect of the corn oil was equally effective against both endogenously released and exogenously injected gastrin. The inhibition occurring in the experiments in which the pyloric gland area pouch was irrigated could be adequately accounted for by inhibitor action against the circulating gastrin released from the pouch. It, therefore, seems unnecessary to implicate an inhibitor action which interferes with the release of gastrin from the pyloric gland area. Of course, the experiments do not exclude this latter possibility as an additional mechanism, but the virtually identical pattern of inhibition seen in the two groups of experiments with regard to both degree and duration, suggests that the same mechanism is active in both instances. It is interesting that the histamine-stimulated responses were not significantly inhibited by feeding corn oil. Although slight inhibition had been previously recorded by several workers, including Kosaka and Lim," Gray et al.,6 and Sircus,7 there has been general agreement that the histamine responses have proved much more resistant to the inhibitory influence. On reviewing all of the available experimental evidence, Gregory8 concluded that there was no convincing indication of fatinduced inhibition of histamine responses which could confidently exclude the possibility of nonspecific inhibition resulting from nausea. The finding of the relative resistance of histamine-stimulated responses to inhibition from fat in the upper small intestine is similar to the behavior pattern with inhibition arising from acidification of the duodenum,9. and the possibility arises that the same humoral agent may be released from the mucosa of the proximal small intestine both by fat and by acid. It is noteworthy that the period of inhibition which followed the ingestion of the corn oil was relatively protracted when compared with most other inhibitor patterns such as that from duodenal acidification, or the intravenous injection of other inhibitor agents. A possible explanation is that the delay in gastric emptying!! and possibly in intestinal transit may have caused -a prolonged contact between the fat and the mucosa of the upper small intestine. REFERENCES 1. Feng, T. P., H. C. Hou, and R K S. Lim. 1929. On the mechanism of the inhibition of gastric secretion by fat. Chin. J. Physiol. 3: 371-380. 2. Kosaka, T., and R K S. Lim. 1930. Demonstration of the humoral agent in fat inhibition of gastric secretion. Proc. Soc. Exp. Bioi. 27: 8-891. 3. Gregory, R A., and H. J. Tracy. 1959. The action of enterogastrone on gastric secretion. J. Physiol. (London) 149: 58-59. 4. Gillespie, L E., and M. I. Grossman. 1963. In Some experiments with gastrin extracts. M. D. Thesis of L E. Gillespie, Glasgow University. 5. Shay, H., J. Gershon Cohen, and S. S. Fels. 1939. The role of the upper small intestine in the control of gastric secretion; the effect of neutral fat, fatty acid and soups; the phase of gastric secretion influenced and the relative importance of the psychic and clinical phases. Ann. Intern. Med. (Chicago) 13: 294-307. 6. Gray, J. S., W. B. Bradley, and A. C. Ivy. 1937. On the preparation and biological assay of enterogastrone. Amer. J. Physiol. 118: 463-476. 7. Sircus, W. 1958. Studies on the mechanisms in the duodenum inhibiting gastric secretion. Quart. J. Exp. Physiol. 43: 114-133. 8. Gregory, R A. 1962. Secretory mechanisms of the gastro-intestinal tract, p. 117-133. Edward Arnold and Co., London. 9. Andersson, S. 1960. Inhibitory effects of HCl in the duodenum on gastrin-stimulated gastric secretion in Heidenhain pouch dogs. Acta Physiol. Scand. 50: 5-112.. Andersson, S. 1960. Inhibitory effect of HCl in antrum and duodenum on histamine-stimulated gastric secretion in Pavlov and Heidenhain pouch dogs. Acta Physiol. Scand. 50: 186-196. 11. Farrell, J. I., and A. C. Ivy. 1926. Demonstration of a dog with a transplanted gastric pouch and of a dog with a pancreatic transplant. Amer. J. Physiol. 76: 226.