stimulating effect on gastric secretion. This view was based on the experiments Thiry-Vella loop.

Transcription

1 418 Keohane and Metcalf STRUGGER, S. (1949). Fluoremzenmikromkopie und Mikrobiologie. Hanover: Schaper. THORELL, B. (1945). Nordisk Medicin, 28, WEISBERGER, S., GUYTON,R. A., HEINLE, R. W. and STORAASLI, J. P. (1951). Blood, 6,919. WHALEN, B. C. and WIDDICOMBE, J. G. (1956). J. Physiol. 132, 41P. WISEMAN, B. K. (1932). Folia Hcemat. 46, 346. YOFFEY, J. M. (1936). J. Anat. 70, 507. YOFFEY, J. M. and COURTICE, F. C. (1956). Lymphatics, Lymph and Lymphoid Tissue. London: Edward Arnold & Co. YOFFEY, J. M. and DRINKER, C. K. (1939). Anat. Ree. 73, 417. THE EFFECT OF OLIVE OIL AND OLEIC ACID ON GASTRIC SECRETION IN THE RAT. By R. SCHNEIDER. From the Department of Medical Biochemistry and Pharmacology, The Medical School, Edgbaston, Birmingham, 15. (Received for publication 21st May 1958) In the rat the inhibitory effect of olive oil on spontaneous gastric secretion is weak and erratic. Large amounts of oil have to be used. Previous hydrolysis of the oil markedly enhances the inhibitory effect. Oleic acid and sodium oleate are potent inhibitors of both spontaneous and insulin-stimulated gastric secretion. Draining the upper intestinal juices reduces, but does not abolish, the inhibitory effect of sodium oleate on insulin-stimulated gastric secretion. Distension of the duodenum with liquid paraffin reduces spontaneous as well as insulinstimulated gastric secretion. Distension of the jejunum or ileum does not have this effect. Distension of the duodenum with sodium oleate as well as with water reduced gastric secretion. There is a positive correlation between the inhibitory effect and the distending pressure. At the same pressure sodium oleate is more potent in depressing gastric secretion than is water. THE inhibitory effect of fat on gastric secretion has been known since the classical experiments of Beaumont [1838] and Ewald and Boas [1886]. Further experimental work by Szokolov [1904] and Lonnquist [1906] showed that the effect was due to contact of fat with the mucosa of the small intestine. Products of lipolysis such as fatty acids and soaps were supposed to have a stimulating effect on gastric secretion. This view was based on the experiments of Piontkowsky [1904] and Babkin [1905], who showed that the introduction of sodium oleate through the duodenal fistula of a dog caused secretion from "the little stomach", and on the experiments of Ivy and Mcllvain [1923-4], who obtained secretion from dogs with fundic pouches after the introduction of short chain fatty acids and "Ivory Soap" into a Thiry-Vella loop. This generally accepted view was first challenged by Roberts [1931] and Shay, Gershon-Cohen and Fels [1939]. Roberts showed in experiments on man that not only neutral fat, but also free fatty acids, inhibited gastric secretion; Shay, Gershon-Cohen and Fels-also in man-produced marked depression of secretion during and after the intraduodenal instillation of a solution of sodium oleate. Recently Sircus [1958] working with dogs obtained a depression of gastric secretion with the intraduodenal instillation of olive oil incubated with pancreatic juice.

2 Fat and Gastric Secretion 419 Experiments on dogs [Quigley and Meschan, 1941] and man [Card, 1941] showed that soaps and free fatty acids also depressed gastric motility, and that the products of lipolysis were often more potent inhibitors than the corresponding neutral fat. Clinical data also point to the greater potency of the products of lipolysis in depressing gastric function. Both in tropical sprue and idiopathic steatorrhcea depression of gastric secretion is a frequent if erratic symptom [v.d. Scheer, 1905; Baumgartner and Smith, 1927; Serra, 1929; Fairley, 1930; Hess Thaysen, 1932; Frazer, 1950] and according to Frazer [1949] decreased gastric motility also is a marked feature of the sprue syndrome. In this syndrome particulate absorption of triglycerides is at fault, and abnormal quantities of fatty acids may occur in the intestine [Frazer, 1950]. The present investigation was undertaken in order to determine the relative potency of neutral fat and certain products of lipolysis in inhibiting gastric secretion in the rat. METHODS Operative Procedures.-White male rats weighing g. were used. The night before the experiment all solid food was removed and the rats given a 5 per cent glucose solution to drink. In some of the experimental series 2- per cent casein hydrolysate was added to the glucose solution. On the day of the experiment a laparotomy was performed under ether anaesthesia, the stomach exposed and a small incision made just proximal to the pylorus. Great care was taken not to injure the blood vessels during this procedure. Two glass cannulae of 2 mm. internal and 3 mm. external diameter, with a rectangular bend, were introduced through this incision and firmly secured by ligatures. One cannula was introduced caudally through the pylorus into the duodenum, the other pointing orally into the stomach. Both cannule carried a length of rubber " drainage " tubing of 1 mm. internal diameter. The tubing on the duodenal cannula was about 45 cm. in length. The distal end was tied, and the whole tubing and cannula filled with the substance under investigation. The tube on the gastric cannula was about 1 cm. long and left open. The purpose of the gastric cannula was to allow the stomach to be emptied at the beginning of the experiment and any food residue to be washed out. The abdominal incision was closed with a continuous suture and the wound painted with celloidin. The tubing of the gastric cannula was then closed with a small artery clip (fig. 1). The rats were put into single wire cages with wire bottoms to prevent coprophagy. The closed end of the duodenal tube was taken through the top of the cage and secured with an artery clip. The rats recovered from the aneesthetic after a few minutes and were able to move about the cage. All injections were given into the duodenal tube without disturbing the rats. At the end of the experiment the rats were sacrificed, the stomach contents aspirated, the volume measured, the ph determined with indicator papers, the free acid titrated with N/100 NaOH, using methyl orange as indicator, and the total chlorides estimated according to Patterson's micro-technique [Harrison, 1943]. In some of the experiments the volume of the small intestine from pylorus to ileocaecal valve was measured and the ph again determined with indicator papers. In a few of the experiments rats were fitted with a duodenal fistula in order to drain the upper intestinal secretions. A glass cannula of the same design as that used for the stomach was introduced through a small incision in the anti-mesenteric border of the duodenum well distal to the entry of the bile duct and firmly tied in. A glass reservoir of about 3 ml. capacity was attached to the cannula with rubber tubing so VOL. XTIII., NO

3 420 Schneider that the drained fluid could be collected. A second cannula was introduced immediately distal to the first, through which the substances under investigation could be administered in the usual way. Method for obtaining a Controlled Fluid Pressure.-A ligature was tied round the duodeno-jejunal flexure. A duodenal cannula was introduced through the pylorus and connected with a glass reservoir of 7 mm. diameter. The whole system was completely filled with the material under investigation, and the top of the reservoir set at the required height above the bottom of the cage. Any increase of intraduodenal pressure was compensated by the spilling of fluid over the top of the reservoir and any reduction of pressure by refilling the reservoir to the original level. l l 3" 1' Top-of cage. Pylorus. Stomach. -Gastric cannula. Duodenum. Duodenal cannula. FIG. 1.-Diagram of the operative procedure. Hydrolysis of Olive Oil.-Olive oil buffered at a ph of 7-8 was shaken at 370 C. with bile salts and pancreatin for hr., and the resultant hydrolysate extracted with ether. The ether was evaporated and the residue used. An analysis of the hydrolyzed olive oil showed the following composition: triglyceride 18 per cent, diglyceride 10 per cent, monoglyceride 10 per cent, free acid 62 per cent. RESIULTS The Effect of Olive Oil on Spontaneous Gastric Secretion.-Unemulsified undiluted olive oil (BP) was introduced intraduodenally in amounts of 45 mg. every 20 min. over a period of 6 hr. The control animals received the corresponding amount of water. The results are shown in Table I. Under these conditions olive oil did not have any inhibitory effect on gastric secretion. On the contrary, the mean values for both free acid and total chlorides were raised. The difference between the means of controls and tests, however, was not significant at the 0 05 level. The amount of olive oil was therefore increased. Only when 180 mg. were used a significant depression of secretion resulted.

4 Fat and Gastric Secretion After previous hydrolysis olive oil became a potent inhibitor of gastric secretion. Amounts as small as mg. caused a significant depression of free acid as well as total chloride secretion. TABLE I.-EFFECT OF UNHYDROLYZED AND PREHYDROLYZED OLIVE OIL ON SPONTANEOUS GASTRIC SECRETION Duration of experiment: 6 hr. Amount in No. of Mean total Mean total Substance mountm chloride in free HCI in S.E. t P mg*/2 mm.rats m.equiv. m.equiv. Controls (water ml.) ±0 066 Olive oil ± *3 6 0*690 ± * ± < 0* ± *100 < Prehydrolyzed olive oil '980 < ± *300 < The Effect of Oleic Acid and Sodium Oleate on Spontaneous Gastric Secretion.-As olive oil after hydrolysis had an inhibitory effect, oleic acid, its main constituent fatty acid, was administered under the same conditions. Oleic acid was used both in the free form and as the sodium soap in a 10 per cent solution. The results are shown in Table II. With oleic acid depression TABLE I.-EFFECT OF OLEIC ACID AND SODIUM OLEATE ON SPONTANEOUS SECRETION Duration of experiment: 6 hr. Amount in No. of Mean total Mean total Substance mg./20 min. rats chloride in free HCI in S.E. t P m.equiv. m.equiv. Controls (water ml.) ±0-066 Oleic acid ± ' ± *930 0* ± < ± < Sodium oleate ± < ± < ± *250 < ± < of both free acid and chloride was obtained with amounts as small as mg., and an increase of the dose to 45 mg. completely suppressed acid secretion. The sodium soap in even smaller amounts (5-10 mg.) depressed secretion and in amounts of 15 mg. or over caused complete suppression. The Effect of Oleic Acid and Sodium Oleate on the Secretory Response to Insulin-induced Hypoglyca-mia.-The results are shown in Table III. The amount of insulin used was 0-15 unit/100 g. body weight. With this amount toxic symptoms were avoided and the secretory response of the stomach after 3 hr. was of the same order as the spontaneous secretion after 6 hrs. Both

5 422 Schneider sodium oleate 20 mg. and oleic acid 45 mg., given every 20 min., again produced a significant depression of both chloride and free acid secretion. TABLE III.-THE EFFECT OF OLEIC ACID AND SODIUM OLEATE ON GASTRIC SECRETION STIMULATED BY INSULIN INDUCED HYPOGLYCAEMIA Duration of experiment: 3 hr. Mean total Mean total Substance 0Amount in No of chloride in free HCI in S.E. t P m.equiv. m.equiv. Controls (water ml.) ± *53 ±0-08 Oleic acid ± ± *02-0*01 Sodium oleate ± < ± < The Effect of Draining the Upper Intestinal Juices on the Inhibition of Gastric Secretion by Sodium Oleate.-The same experimental procedures were carried out on rats that had been fitted with duodenal fistulae in order to study the importance of the upper intestinal juices on the inhibitory mechanism. This time sodium oleate was used and only free acid determined. The results are shown in Table IV. Although the intraduodenal administration of sodium oleate still caused inhibition of gastric secretion, the degree of depression compared with that in the animals without duodenal fistulke had been significantly reduced (t: 3-1; P: ). TABLE IV.-THE EFFECT OF SODIUM OLEATE ON GASTRIC SECRETION STIMULATED BY INSULIN INDUCED HYPOGLYCAXMIA Duration of experiment: 3 hr. Amount i No. of Type of Mean total Substance mg./20 Amount min. in No. rats of preparation Type of free HCI in S.E. t P m.equiv. Controls (water) Duodenal fistula ±0-089 Sodium oleate Duodenal fistula ± The Effect on Gastric Secretion of Distending the Small Intestine with Liquid Paraffin.-In order to assess what effect distension of the small intestine alone might have on gastric secretion, liquid paraffin was introduced intraduodenally in a single amount of 5 ml. and a ligature tied immediately beyond the material administered. The effect was studied on spontaneous secretion (fig. 2) as well as on insulin stimulated secretion (Table V). Spontaneous secretion was completely suppressed, and the response to insulin induced hypoglyceemia significantly reduced. When the site of administration was changed to the middle of the jejunum, or to the more distal portion of the ileum, no inhibition of gastric secretion resulted (Table V). Comparison of the Inhibitory Effect of Olive Oil on Gastric Secretion Stimulated by Insulin-induced Hypoglyce3mia with that of Liquid Paraffln.-In this series of experiments olive oil and liquid paraffin were administered intra-

6 Fat and Gastric Secretion duodenally in equal amounts. The rats were fitted with duodenal fistulae to avoid the effect of the additional volume of upper intestinal juices. The amount of oil was 270 mg. every 20 min. for 3 hr. and insulin-induced hypoglycaemia was again used as the stimulus for gastric secretion. The results FIG. 2.-The effect of the intra- 8 duodenal administration of a single dose of liquid paraffin on 7 spontaneous gastric secretion in conscious rats. Pylorus and *6 duodenum ligated. Duration //: _ of experiment 6 hr. 5 %/R The hatched columns represent.4 t/ the means of total chlorides and.*4 /7 the clear columns the means of e A/ the free acid secreted. The bars a' *3-0represent the standard errors of the means. The numbers refer 2 to the numbers of animals used. L.P. =liquid paraffin. IiB ~~4 4 Controls L.P. 5 ml. 423 with olive oil were erratic. Complete or almost complete depression of free acid secretion occurred with three of the six rats examined. With liquid paraffin depression of secretion occurred in four of the six rats used and there was no significant difference between the means (t: 0.32). Doubling the amount of liquid paraffin did not enhance its inhibitory effect. TABLE V.-THE EFFECT OF DISTENSION WITH A SINGLE DOSE OF 5 ML. OF LIQUID PARAFFIN ON GASTRIC SECRETION STIMULATED BY INSULIN INDUCED HYPOGLYCAEMIA. Duration of experiment: 3 hr. Mean total Mean total Site of No. of chloride in free HCI in S.E. t P distension rats m.equiv. m.equiv. Controls (no distension) ±0-08 Duodenum ± < ± <0.001 Jejunum or ileum ± ± The Effect of Pressure Changes in the Duodenum on the Inhibitory Effect of Distension on Gastric Secretion. (Comparison of the Effect of Sodium Oleate with that of Water.) In these experiments a ligature was tied around the duodeno-jejunal flexure, the duodenum was distended with sodium oleate and the distending pressure kept constant throughout the experimental period of 3 hr. Insulin-induced hypoglycwlmia was used as the stimulus for gastric secretion. Fig. 3a shows a significant correlation between the distending pressure and the inhibition of gastric secretion (r: ; P: ). When water was used instead of sodium oleate (fig. 3b) a

7 424 Schneider significant correlation (r: -0O516; P: ) was again found, but water was less potent in depressing gastric secretion than sodium oleate. 10 * r= *9 P *8-7 ~ 7 E6 0 I 4 ow Q 1O 30 Pressure in crn.(logscale.) (a) r= *1 P= =.9 w.7- %J.4. o2* I 4-.3.~ ~ ~ :> 7 Pressure in 10 cm.(log, scale) (b) FiG. 3.-The effect of intraduodenal pressure on gastric secretion of free HCl by conscious rats. The pressure is expressed as the height of the fluid level above the wire bottom of the cage (log. scale). r: correlation coefficient. The lines are calculated regression lines. (a) the effect of 10 per cent sodium oleate. the effect of water. (b) DISCUSSION Piontkowsky [1904] and Babkin [1905], and recently Sircus [1958] administered fatty acid intraduodenally into dogs and found a stimulation of gastric secretion. The reason for the difference in the two species is not clear. Unfortunately, neither the concentrations of the solutions nor of the exact position of the fistulse are stated by the earlier workers. A possible stimulating effect of weaker concentrations would be more easily detected in dogs,

8 Fat and Gastric Secretion who frequently have no fasting gastric secretion [Babkin, 1950; Necheles, 1955] than in rats, who secrete large amounts of acid gastric juice when fasting. Since the experiments of the Pavlov school [Szokolov, 1904 and Lonnquist, 1906] it has been accepted that the inhibitory effect of fat on gastric secretion is due to its presence in the intestine. During the present investigation it was found that inhibition of gastric secretion was frequently associated with the accumulation of large amounts of fluid in the intestine at the end of the experimental period. This suggests that the upper intestinal juices might play a part in the inhibitory mechanism. Draining the upper intestinal juices significantly reduced, but did not abolish, the inhibitory effect of sodium oleate. Under these conditions the volume of the intestinal fluid found at the end of the experiment was reduced, but was still larger than that of the control animals. These findings suggest that distension of the intestine with fluid might be an important factor in the inhibitory mechanism of fat on gastric secretion. The inhibitory effect of distension of the upper small intestine on gastric secretion in dogs was first mentioned by Day and Webster [1935] and later shown to vary with the degree of the distending pressure [Sircus, 1953; Walker, 1955]. It is well known that hypertonic solutions of glucose are potent inhibitors of gastric secretion in man and dog [Muir, 1949], and it seems likely that the superiority of intraduodenal hypertonic glucose over that of intravenous glucose as an inhibitor of gastric secretion [Muir, 1949] is due to the duodenal distension it evokes. In an attempt to demonstrate the effect of distension on gastric secretion, unemulsified liquid paraffin was introduced intraduodenally in the present investigation and prevented from passing down the intestine by a ligature just beyond the material administered. A significant inhibition of both spontaneous secretion and insulin stimulated secretion was found. With changing the site of administration to the jejunum or the ileum, no depression of gastric secretion occurred. These findings suggested that the inhibitory mechanism operated from the duodenum only and not from the more distal portions of the small intestine. If the distending material was allowed to pass down the intestine, depression of gastric secretion still occurred, but the results became erratic. There was no difference between the inhibiting effect of olive oil and liquid paraffin under these conditions. The fact that increasing the volume of the single dose of distending material from 0 3 ml. to 0X6 ml. did not increase the effect indicated that the distending pressure in the duodenum was a more important factor in this mechanism than the volume of fluid administered. This was confirmed by the finding of a negative correlation between distending pressure in the duodenum and the degree of free acid secretion by the stomach. Such a negative correlation was found with water, as well as with sodium oleate. Sodium oleate, however, seemed to inhibit gastric secretion more than water at the same distending pressure. In the sprue syndrome several factors contribute to cause distension of the small intestine. Absorption is generally depressed and increased amounts of free fatty acids may be present in the intestine. Further small intestinal 425

9 .426 Schneider motility is reduced and there is excess mucus secretion; also an increase of volatile fatty acids, due to fermentation, is a frequent occurrence. The radiological picture of the distended small intestine after the administration of non-flocculating barium is one of the characteristic features of the syndrome. The present findings in the rat suggest that such distension of the duodenum might be a major factor in producing hypochlorhydria or achlorhydria as they frequently occur in the sprue syndrome, and might also account for the erratic nature and the reversibility of these symptoms. ACKNOWLEDGMENTS This work was started during the tenure of a Leverhulme Research Scholarship, and the author wishes to thank the foundation for financial assistance. His thanks are also due to Professor A. C. Frazer and Dr. H. G. Sammons for their valuable help and constructive criticism, to Dr. J. A. H. Waterhouse for advice regarding the presentation of the statistical data, and to his technical assistants, mainly Mrs. H. Bishop and Mr. A. G. Whyborn, for their untiring help. REFERENCES BABKIN, B. P. (1905). BABKIN, B. P. (1950). Arch. Sci. biol., St. Petersb. 11, 214. Secretory Mechanism of the Digestive Glands. 2nd Ed. New York: Paul B. Hoeber, Inc. BAUMGARTNER, A. and SMITH, G. D. (1927). Arch. intern. Med. 40, 203. BEAUMONT, W. (1838). Experiments and Observations on the Gastric Juice. Edinburgh. CARD, W. I. (1941). Amer. J. dig. Dis. 8, 47. DAY, J. J. and WEBSTER, D. R. (1935-6). Amer. J. dig. Dis. 2, 527. EWALD, C. A. and BOAS, J. (1886). Virchows Arch. 104, 271. FAIRLEY, H. N. (1930). Trans. B. Soc. trop. Med. Hyg. 24, 131. FRAZER, A. C. (1949). Brit. med. J. ii, 47. FRAZER, A. C. (1950). Fat Absorption and the Sprue Syndrome. Presented 1950 to the Basic Sciences Course, Army Medical Service Graduate School, Walter Reed Army Medical Center. HEss THAYSEN, TH. E. (1932). Non-tropical Sprue. A Study in Idiopathic Steatorrhcea. Copenhagen: Levin and Munksgaard. London: Oxford University Press. Ivy, A. C. and MCILVAIN, G. B. (1923-4). Amer. J. Physiol , 124. LoNNQuIsT, B. (1906). Skand. Arch. Physiol. 18, 194. MUIR, A. (1949). Quart. J. Med. 18, 235. NECHELES, H. (1955). Department of Gastro-intestinal Research, Medical Research Institute of Michael Reese Hospital, Annual Report for the year PIONTKOWSKY, L. F. (1904). Proceedings of the Ninth Pirogovski Congress, 1, part 3, 10th January. QUIGLEY, J. P. and MESCHAN, I. (1941). Amer. J. Physiol. 134, 806. ROBERTS, W. M. (1931). Quart. J. Med. 24, 133. V.D. SCHEER, A. (1905). Mense Handbuch der Tropenkrankheiten. Leipzig: I. A. Barth. V.D. SCHEER, A. (1905). Ned. Tijdschr. Geneesk. 41, 637. SERRA, A. (1929). Amer. J. trop. Med. 9, 49. SHAY, H., GERSHON-COHEN, J. and FELS, S. S. (1939). Ann. intern. Med. 131, 294. SIRCUS, W. (1953). Quart. J. exp. Physiol. 38, 91. SIRCUS, W. (1958). Quart. J. exp. Physiol. 43, 114. SZOKOLov, A. (1904). Jber. Fortschr. Tierchem. 34, 469.