GASTROENTEROLOGY Copyright 1967 by The Williams & Wilkins Co. Vol. 52, No.3 Printed in U.S.A. RELEASE OF HISTAMINE INTO GASTRIC VENOUS BLOOD FOLLOWING INJURY BY ACETIC OR SALICYLIC ACID LEONARD R. JOHNSON AND BERGEIN F. OVERHOLT, M.D. Department of Physiology, University of Michigan, Ann Arbor, Michigan It has frequently been suggested! 2 that histamine is liberated within the gastric mucosa during stressful or damaging situations. Davenport has shown that, when the mucosa is injured by salicylic or acetic acid, a number of changes occur,3-5 some of which could be caused by an intramucosal liberation of histamine. The flow of fluid from the damaged mucosa exceeds that of control experiments, indicating increased capillary permeability as well as increased mucosal permeability. In addition, plasma proteins appear in the gastric contents in excess of erythrocytes, which is evidence that the volume changes are not primarily caused by bleeding into the lumen. Whether acid secretion increases as well as volume flow has not been determined, since hydrogen ions move through the damaged mucosa toward the serosal side and are neutralized by HC03 ions in ~he mucosal extracellular fluid which enters the lumen. Topical irrigation of the damaged mucosa with histamine produces a model of the effects of histamine release. There is a large increment in volume, most but not all of which is plasma. If acid secretion is trapped with Received August 1, 1966. Accepted October 31, 1966. Address requests for reprints to: Leonard R. Johnson, Department of Physiology, East Medical Building, University of Michigan, Ann Arbor, Michigan 48014. This investigation was supported by Research Grant AM-08716 from the United States Public Health Service. L. R. Johnson was supported by a Predoctoral Fellowship from the United States Public Health Service. The authors are grateful to Hazel Brumby, Virginia D. Davenport, and Inez Mason for expert technical assistance and to Dr. H. W. Davenport for continued advice and support. 505 glycine buffer and if the parietal cells secrete acid at a concentration of 150 mm, Davenport has calculated that secretion accounts for approximately 11% of the volume increment during histamine irrigation and for 65% of the increment during the next 30 min. Histamine applied to the normal mucosa has no effect. Tentative evidence for the intramucosal liberation of histamine during damage was provided by the observation that histamine appeared in Heidenhain pouch contents during and following salicylate damage more often than in control experiments.s Further evidence comes from work with the pylorus-ligated rat. We have shown that, when the rat gastric mucosa is irrigated with 100 mm acetic acid or 20 mm salicylic acid in 100 mm HCI, histamine appears in the gastric contents and the histamine content of the oxyntic glandular area simultaneously decreases.7 These changes do not occur when the stomachs are filled with 154 mm N aci or with 100 mm HCl. We inferred from this that, during injury, histamine is.present in the mucosal interstitial fluid where it can exert its characteristic effects. Unequivocal evidence for histamine liberation within the mucosa would be the finding of increased levels of histamine in the interstitial fluid during injury. Appearance of histamine in gastric venous blood or lymph would be presumptive evidence of its presence in the interstitial fluid. The present report concerns an investigation of the histamine content of gastric venous blood during injury. Materials and Methods Fifteen dogs weighing 15 to 22 kg were fasted overnight and then anesthetized with sodium pentobarbital, administered intravenously. The
506 JOHNSON AND OVERHOLT Vol. 52, No.3 Left gostroepi 'pio' IC vein *,,~----;:,plerllc vein Blood collecting --t':.--...!-~~ catheter FIG. 1. Diagram of method used to isolate and collect gastric venous blood. spleen was delivered through a midline abdominal incision, and a catheter was inserted into one of the splenic veins (fig. 1). Following splenectomy, a.length of umbilical tape was passed under the gastrosplenic vein between its junctions with the portal and left gastric veins. During collection of venous blood, the tape was pulled, occluding the gastrosplenic vein and causing a retrograde flow of gastric venous blood from the catheter. Normal blood flow resumed when the umbilical tape was released. A tube was then passed into the stomach through an incision in the duodenum and tied in place, as illustrated in figure 1. The stomach was filled with 300 ml of one of four solutions: (1) 154 mm NaCI, (2) 100 mm acetic acid, 15 mm NaCI, 78 mm mannitol, (3) 100 mm HCI, 15 mm NaCI, 78 mm mannitol, 20 mm salicylic acid, (4) 100 mm HCI, 15 mm NaCI, 78 mm mannitol. Just prior to irrigation and at various times thereafter, 5-ml gastric venous and femoral arterial blood samples were collected. Blood samples were immediately combined with 10 ml of 10% w Iv trichloroacetic acid, mixed, and filtered as described by Code. Samples of the irrigation solutions were also removed from the stomachs for histamine assay. The trichloroacetic acid filtrates of the blood and the samples of irrigation fluid were combined with 5 ml of concentrated HCI to begin the histamine extraction procedure described by Code and McIntire. Histamine content of the final extracts of both blood and fluid was determined by assay using an isolated terminal segment of guinea pig ileum suspended in atropinized Tyrode's solution: Potassium concentrations of the final extracts were measured by internal standard flame photometry and found to be well below the level which would affect the isolated ileum. ll Contractions attributed to histamine were abolished by 1.0 mg per liter of pyrilamine maleate. All results are expressed as micrograms of histamine base. The minimal amount of detectable histamine was 1.2 p.g per liter of blood or gastric contents, and quantities below this are recorded as zero. This method recovers total histamine present in the blood. In each of the 15 dogs, two arterial and two gastric venous blood samples were assayed for histamine before filling the stomachs. The four values were always essentially the same in each dog and ranged from 0.0 p.g per liter to
March 1967 HISTAMINE IN GASTRIC VENOUS BLOOD 507 56.8 flg per liter in the 15 dogs, with a mean of 6.4 flg per liter. The second highest concentration was 20.3 flg per liter. Recoveries of histamine added to blood samples varied from 62 to 100%. The errors of replication in duplicate determinations ranged from 2 to 17%. 10,-------,----.----,------,-- --~---" Results Histamine Levels in Gastric Venous Blood NaCl and HCl Instillations. In five stomachs filled for 120 min with the N aci solution and three filled with the 100 mm HCI solution, there was no increase in arterial or gastric venous histamine over preinstillation levels. Acetic acid instillations. During exposure to acetic acid, histamine appeared in the gastric venous blood within 5 min, and the increase over pre instillation levels was 6.8 to 9.0 flg per liter (fig. 2). In the four acetic acid experiments, histamine concentration dropped within 10 or 15 min, returning to control levels by 45 min. Arterial samples taken at the same time as the venous samples containing the peak concentrations of histamine had only a fraction of the venous histamine or none at all. Before filling, arteriovenous differences were not discernible in any dog having detectable blood histamine at that time, and, at the o 5 10 15 30 Time (minutes) FIG. 2. Change in the histamine concentration of gastric venous blood during acetic acid irrigations. The amount of histamine present during the control period of an experiment was subtracted from that present at each time interval following instillation of acetic acid at zero time. These values were averaged for each time interval. Vertical lines indicate standard errors of the means. The increase over the control level is significant with P < 0.01. Time {minutes} FIG. 3. Change in the histamine concentration of gastric venous blood during salicylic acid irrigations. The amount of histamine present during the control period of an experiment was subtracted from that present at each time interval following instillation of salicylic acid at zero time. These values were averaged for each time interval. Vertical lines indicate standard errors of the means. The increase over the control level is significant with P < 0.05. end of a 60-min irrigation, there were again no detectable arteriovenous differences. Salicylic acid instillations. In the three salicylic acid experiments (fig. 3), hi~tamine appeared in the gastric venous blood more slowly; the maximum postinstillation difference occurred at 15 min, the range being 4.5 to 9.8 flg per liter. As in the acetic acid experiments, blood histamine fell rapidly after 15 min and reached near control levels by 60 min. Again there were no arteriovenous differences at the beginnings and ends of the irrigations and no or only slight increases in' arterial histamine during the increases in venous levels. Histamine Levels in Gastric Contents N acl and HCl Instillations. In four NaCI and three HCI experiments, histamine was absent from samples of the solution removed from the stomach until 60 min had elapsed. In one experiment, low amounts of histamine were found at the beginning. The highest concentration found was only 3.1 flg per liter. Acetic and salicylic acid instillations. Histamine, however, appeared in all acetic acid solutions (fig. 4) within 5 min. The range at 5 min was 2.5 to 9.0 flg per liter. All salicylic acid solutions also contained
508 JOHNSON AND OVERHOLT Vol. 62, No.3 Noel 30 45 60 75 90 105 120 Time of Irrigotion (minutes) FIG. 4. Comparison of amounts of histamine liberated into the lumens of stomachs filled with N aci versus those filled with acetic acid. Means and standard errors of the means at various time intervals are shown. histamine within 5 min, the range being 5.4 to 39.1 p.g per liter. Discussion A summary of our concept of the role of histamine in explaining a portion of the results observed during damage to the gastric mucosa has been presented by Davenport.6 The acetic and salicylic acid are present in the lumen as almost 100% undissociated and lipid soluble molecules. As such they diffuse across the mucosa and ionize at the higher intracellular ph. The released H + damages the tissue, and acid diffuses through the mucosa, made much more permeable by the injury. The arid causes a liberation of histamine into the interstitial fluid. If the mucosa is permeable enough, some histamine appears in the gastric contents. Some histamine stimulates secretion and some increases mucosal capillary permeability, resulting 111 the presence of an abnormal amount of plasma proteins in the gastric contents. It is difficult to assess the amounts of histamine needed to cause these changes, especially since all of the histamine is released from the gastric mucosa and can be expected to have a greater effect on the mucosa than the same amount originating somewhere else. The increase of approximately 9 p.g per liter observed in the gastric venous blood in these experiments would seem to be more than sufficient to account for the hypothesized changes. In a group of experiments (unpublished data), we have found that intravenous infusion of a low concentration of histamine always produces a significant secretion of hydrogen ion by the oxyntic cells before gastric venous histamine concentrations have increased 5 p.g per liter. The time course of the histamine release into the gastric venous blood supports the general concept of the role histamine plays in damage to the gastric mucosa. In figure 2, following acetic acid injury the venous blood histamine appears quickly and peaks sharply at 5 min. With salicylic acid (fig. 3), there is little histamine present at 5 min, and the peak concentration does not occur until 10 to 15 min. This was predicted from the model, for the diffusion gradient for acetic acid is much greater than for salicylic acid (100 mm versus 20 mm). Therefore, it should take longer for the salicylic acid to diffuse through the mucosal barrier and injure the tissue. There are, however, several interesting problems raised by the time course of release. The release occurs quickly and is of short duration. Our experimental setup does not allow us to differentiate between liberation of preformed histamine and synthesis and release of histamine. Therefore, the pulse of liberated histamine could represent a brief release of histamine from mast cells or a short stimulation of the histamine-forming capacity of the tissue. On the other hand, there is the less likely possibility that histamine is liberated throughout the experiment, but its rate of destruction increases to such an extent that after the first 15 min little escapes into the gastric venous blood. Summary The stomachs of anesthetized dogs were filled with 154 mm N aci, 100 mm HCI, 100 mm acetic acid, or 20 mm salicylic acid in 100 mm HCI. Gastric venous and femoral arterial blood samples were taken at various intervals for the next 120 min, and their histamine content was determined. Within 5 min there was a significant increase in
March 1967 HISTAMINE IN GASTRIC VENOUS BLOOD 509 the histamine concentration of the blood draining the stomachs filled with acetic acid. A similar increase occurred within 10 min in the blood from the salicylic acidtreated stomachs. Femoral arterial samples taken concurrently with the gastric venous samples containing the peak amounts of histamine contained little or no histamine. In no experiment was an increase in histamine detected in blood from a stomach containing NaCl or HCI. Histamine always appeared early in the salicylic and acetic acid irrigation solutions and reached peak concentrations at 15 min. Histamine was usually absent from the NaCl solutions until 1 hr of irrigation had elapsed, and then the concentrations reached were low. We conclude that the increased amount of histamine in the gastric venous blood during injury is proof that it is released into the mucosal interstitial fluid, where it can exert its characteristic physiological actions. REFERENCES 1. Clark, B. B., and W. L. Adams. 1947. The effect of acetylsalicylic acid on gastric secretion. Gastroenterology 9: 461-465. 2. Rasanen, T. 1961. Effect of prednisolone and deoxycorticosterone, administered separately and with growth hormone, on the mucosal mast cells and tissue eosinophils of rat stomach wall. Gastroenterology 40: 232-234. 3. Davenport, H. W. 1964. Gastric mucosal injury by fatty and acetylsalicylic acids. Gastroenterology 46: 245-253. 4. Davenport, H. W. 1965. Damage to the gastric mucosa: effects of salicylates and stimulation. Gastroenterology 49: 189-196. 5. Davenport, H. W. 1965. Potassium fluxes across the resting and stimulated gastric mucosa: injury by salicylic and acetic acids. Gastroenterology 49: 238-245. 6. Davenport, H. W. 1966. Fluid produced by the gastric mucosa during damage by acetic and salicylic acids. Gastroenterology 50: 487-499. 7. Johnson, L. R. 1966. Histamine liberation by gastric mucosa of pylorus-ligated rats damaged by acetic or salicylic acids. Proc. Soc. Exp. BioI. Med. 121: 384-386. 8. Code, C. F. 1937. The quantitative estimation of histamine in the blood. J. Physiol. (London) 89: 257-268. 9. Code, C. F., and F. C. McIntire. 1956. Quantitative determination of histamine, p. 49-95. In D. Glick [ed.], Methods of biochemical analysis, Vol. 3. Interscience Publishers, Inc., New York. 10. Barsoum, G. S., and J. H. Gaddum. 1935. Pharmacological estimation of adenosine and histamine in the blood. J. Physiol. (London) 85: 1-14. 11. Code, C. F., G. A. Hallenbeck, and R. A. Gregory. 1947. Histamine content of canine gastric juice. Amer. J. Physioi. 151: 593-605.