GASTRIC MUCOSAL LESIONS PRODUCED BY INTRAVENOUS INFUSION OF ASPIRIN IN CATS

Transcription

1 GASTRONTROLOGY 71: Copyright <D 197 by The William. & Wilkin. Co. Vol. 71. No.5 Printed in U.S.A. GASTRIC MUCOSAL LSIONS PRODUCD BY INTRAVNOUS INFUSION OF ASPIRIN IN CATS ROLAND BUGAT, MICHAL R. THOMPSON, DOROTHA AURS, AND MORTON I. GROSSMAN VA Wadsworth Hospital Center and University of California at Los Angeles School of Medicine, Los Angeles, California Aspirin was given by continuous intravenous infusion to 35 intact cats for 7 days in doses ranging from 5 to 00 mg kg- I day-i. Gastric mucosal lesions occurred in 50 to 70% of the animals in the various dosage groups, including deep ulcers in 0%. All of the ulcers were in antral mucosa near its border with oxyntic mucosa. The incidence of lesions, including ulcers, showed no apparent relation to the dose of aspirin. With all but the highest dose, plasma salicylate levels were within or below what is regarded as the therapeutic range for man. Aspirin, 100 mg kg- I day- a, was given for 7 days to 4 cats with pouches containing all of the antral mucosa plus some oxyntic mucosa. One or more deep ulcers occurred in the antral mucosa of the pouches in each of these 4 cats. The electrical potential difference across the mucosa did not decrease, and net fluxes of hydrogen ions out of the pouch and of sodium ions into the pouch did not increase during the 7 days of aspirin administration despite the occurrence of ulcers in the pouches. It is concluded that intravenous aspirin, in doses giving plasma levels within or below the therapeutic range for man, causes gastric mucosal lesions including deep ulcers within 7 days in cats. These lesions occur without the changes in electrical potential difference and hydrogen and sodium fluxes that are regarded as characteristic of the "broken barrier." It is well established that aspirin can cause lesions of the gastric mucosa in man and animals. l A widely accepted hypothesis regarding the pathogenesis of these lesions I involves these steps: (1) entry of aspirin into the mucosa from the lumen by nonionic diffusion, () impairment by the absorbed aspirin of the barrier to back diffusion of hydrogen ions (H+) into the mucosa, and (3) back diffusion of H+ into the mucosa leading to injury and lesions. This pathogenetic scheme requires that aspirin be in contact with the m.ucosa from the luminal side. However, it has been known for some time that aspirin given by extragastric routes also can cause gastric lesions.' This study was done to characterize the lesions produced by intravenous infusion of aspirin in cats and Received March 19, 197. Accepted May 10, 197. This study was 8upported in part by Grant AM1738 from the National Institute of Arthritis, Metaboli8m and Digestive Diseases to CUR (Center for Ulcer Research and ducation!. Dr. Grossman's work was supported by a Veterans Administration Senior Medical Investigatorship. Dr. Bugat held a fellowship from the French Ministry of Foreign Affairs. Hi. present address is Centre Claudius Regard. Toulouse, France. Dr. Thompson held a Harkness Fellowship from the Commonwealth Fund. His present address is Department of Surgery, The Royal Infirmary, Manchester, ngland. The authors thank Janet lashoff for statistical analyses, John Washington for technical assistance, and Kuwa Chou for secretarial help. 754 to determine whether they are accompanied by the changes that are commonly used as an index of a "broken barrier," namely increased back diffusion of H+, increased entry of sodium into the luminal fluid, and decreased electrical potential difference (PD) across the gastric mucosa. It was found that intravenous infusion of aspirin in cats produced lesions of the gastric mucosa, varying from erosions to deep ulcers, without the changes that characterize the broken barrier. Methods Aspirin, or saline as a control, was given by continuous intravenous infusion for 7 days to 41 intact cats and to 4 cats with pouches containing all of the antral mucosa and some oxyntic mucosa. Gastric mucosal lesions were looked for in all of these animals. In addition, in the cats with the pouches, studies of ionic fluxes and PD were done. Cats. Mongrel cats of both sexes weighing from.1 to 5.5 kg were used. Antral-oxyntic pouches. The pouches contained all of the antral mucosa plus part of the oxyntic mucosa; they had about equal areas of these two kinds of mucosa. The pouches were drained by plastic cannulas. Gastrointestinal continuity was restored by gastroduodenostomy. Tests were begun 3 or more weeks after surgery. Continuous intravenous infusion for 7 days. ach cat was housed in an individual cage with a volume of about 1 m '. Food (Purina Cat Chow) and water were available at all times. ach cat had a polyethylene catheter (0. mm external diameter) inserted into a foreleg vein and held in place with masking tape. The catheter was connected to a reservoir of infusate by a polyvinyl tube that traversed a peristaltic pump (Harvard

2 November 197 ASPIRIN AND GASTRIC MUCOSAL LSIONS 755 Apparatus Co., Millis, Mass.) that delivered 10 ml per hr. Cats, unlike other species, tolerate prolonged intravenous infusions without restraint and usually do not remove the intravenous catheter. Tissue and blood samples. Food was withheld during the last 1 hr of the 7th day of infusion. Ten minutes before death, 5 ml of 5% pontamine sky blue BX (lcn Pharmaceuticals, Cleveland, Ohio) was given intravenously. When this protein-bound dye leaks through a damaged mucosal surface, it produces a dark blue spot on a pale blue background, allowing lesions as small as 0.3 mm in diameter to be detected.' A sample of blood for measurement of salicylate concentration was taken by cardiac puncture and the animal was then killed by intracardiac injection of sodium pentobarbital. The stomach, including the pouch if it was present, and the small intestine, were removed, opened, and carefully examined grossly for lesions, including those made visible by the dye. A tissue sample of each lesion was fixed in Bouin's solution and histological sections stained with hematoxylin and eosin were made. Samples of antral and oxyntic mucosa uninvolved with lesions and a sample of diaphragmatic muscle were taken for salicylate determination. Ion fluxes and PD in antral-oxyntic pouches. These measurements were made on alternate days for 4 days before starting infusion of aspirin and then each day except the th of the 7 days during the infusion. Food was withheld for 1 hr before each test. The cats were placed in slings on wooden frames.' The cannulas draining the pouches were connected by a polyethylene tube to a three-way stopcock. One port led to a vertical 5O-ml syringe barrel without plunger and the other to a 10-ml syringe used for introducing and removing samples. The residual volumes of the pouches were measured using "CrCI. and ranged from 1.0 to 1.3 ml.' The pouch was washed with Whatever solution was to be tested and allowed to drain for 1 min. The test solution (15 m!) was then instilled and mixed thoroughly with the residual volume, and 5 ml were removed as the initial sample. Thirty minutes later the pouches were emptied and the volume recovered was measured to the nearest 0.1 ml in graduated cylinders. The initial and final samples were analyzed for H+ and Na' concentrations. The initial and final masst's of each constituent analyzed were calculated by ~ u l t i pthe l yinitial i n g and final volumes by the concentrations. The net fluxes were calculated by subtracting the initial from the final masses. Positive values indicate net addition to the pouch and negative values indicate net loss from the pouch. On each test day, four concentrations of HCI-lO, 50, 100, and 150 mm-were tested in random order. The HCl solutions were adjusted to 300 milliosmoles per kg with mannitol. The electrodes used for measurement of PD were polyethylene tubes filled with 3% agar in saturated KCI. One electrode was passed into the pouch through the cannula. The other electrode ended in a hypodermic needle that was placed Subcutaneously. ach electrode led to a beaker containing saturated KCl with a calomel half-cell immersed in it. The Calolllel half-cells were connected to a ph meter (Radiometer, o!>enhagen Denmark) from which PD was read to the nearest Illillivolt. ' Biochemical determinations. In the ion flux studies, titra ~ a b. al ce i dto iph t y 7.0 was measured with an automatic.titrator ;;adlometer) using 0. M NaOH, and Na+ concentration was easured by flame photometry (model 143, Instrumentation!hboratory, Boston, Mass.) The salicylate concentrations of e samples of plasma and tissues were measured by the method of Saltzman using an Aminco-Bowman spectrofluorometer set at 310 n for ~ excitation and 400 nm for emission. SOlution of aspirin for intravenous infusion. A solution of aspirin was freshly prepared each day by dissolving 3 g of NaHCO. and 3 g of acetylsalicylic acid (Sigma Chemical Co., St. Louis, Mo.) in 100 ml of water, giving a solution with a ph of 7.1. This aspirin solution (30 mg per m!) was diluted with sufficient 0.15 M NaCI to give the desired dose rate for each cat when infused at 10 ml per hr. Results Toxic effects. All of the cats getting 00 mg kg-i day- I of aspirin had marked anorexia, losing 30% of their body weight during the 7 days. Four of the cats on this dose of aspirin had repeated convulsions and died on the 4th to 7th days of the test. No convulsions were seen in any of the cats getting doses less than 00 mg kg- I day- I. At 100 mg kg- 1 day- I all of the cats had anorexia by the 5th day, and 1 died on the th day but no cause of death was found at autopsy. The cats getting 5 or 50 mg kg - 1 day- 1 ate normally and maintained their body weight. One cat getting 50 mg kg - ' day- 1 died on the 5th day;. bronchopneumonia was found at autopsy. Salicylate concentrations in blood and tissues. These are summarized in table 1. Plasma salicylate concentration more than doubled with each doubling of the dose of aspirin infused. Similar nonlinearity between dose and blood level has been observed in human subjects and is attributed to a decreased rate of removal as the metabolic pathways for removal of salicylate become saturated. The plasma salicylate levels with the 5 and 50 mg kg - 1 day-' doses were below what is regarded as the therapeutic dose range for man, 150 to 300 Ilg per mi. The concentration of salicylate in tissue was always less than one-half that of plasma. The salicylate concentrations in diaphragmatic muscle, antral mucosa, and oxyntic mucosa were not significantly different. Lesions of the gastric mucosa. Details of the number and location of lesions in individual animals are given in table and a summary of incidence in relation to dose of aspirin is given in table 3. Lesions that extended through the muscularis mucosae are designated as ulcers. No lesions occurred in the cats that received saline intravenously for 7 days. The incidence of lesions varied from 50 to 70% in the various dosage groups, an insignificant difference (P > 0., Fisher's exact test for difference between the group with the highest incidence and the other three groups combined). By a similar test there was no significant difference in the incidence of ulcers in the various dosage groups. The only statistically significant difference attributable to dosage was in the number of lesions per stomach. Five of 10 cats in the TABL 1. Mean (±S) concentration of salicylate in plasma (micrograms per milliliter) and in tissues (micrograms per gram) No. of A si rp m d o ~ C 8 t ~ mll ~ " R day ' I Plasma Concentration of s.allcylate O x ~ m t i Antral c mucosa mucosa 3±5 3±44±3 4±5 90 ± 13 4 ± 4 43 ± 4 30 ± ± ± 3 94 ± ± 4 D i a p h r a ~ m

3 75 BUGAT TAL. Vol. 71, No.5 00 mg kg I day - I group had more than six lesions in their stomachs compared to only 1 of the 4 cats in the other three dosage groups combined; this difference IS significant at P < 0.05, Fisher's exact test. TABL. Lesions in intact cats that received a continuous intravenous infusion of aspirin for 7 days ('at no. 4.'i 7 ~ 9 10 II lfi 17 I ~ ;> Aspirin dose ;' :!!j!) :!f) ;) ;) ~ 5 :!f),io f,o,,0,.0,,0 ~ O 1(1) lin) lin) IIX) 11K) I(x) IIlIl IOIJ IOIJ OX\,lltl!" ni(''; during glanri Antrum DllooellulIl l'xpnltlll'nt :; 4.) :; 1 The commonest location of lesions was in antral mucosa near its junction with oxyntic mucosa (fig. 1). All of the ulcers and many of the more superficial lesions were in this location. The lesions of the oxyntic gland area often were situated on the crest of a mucosal fold (fig. ). Microscopically, all of the lesions, including ulcers, showed only minimal inflammatory reaction. The bases of the ulcers showed a moderate degree of fibrosis (fig. 3). No histological changes were found in the mucosa uninvolved in grossly visible lesions. All 4 of the cats with antral-oxyntic pouches that got 100 mg kg - I day I of aspirin for 7 days had one or more deep ulcers in the antral mucosa as well as numerous smaller and more superficial lesions, particularly of the oxyntic mucosa (figs. 4 and 5). Ion fluxes and PD in antral-oxyntic pouches. The PD and ion f1uxes did not change significantly during the 7 days of aspirin infusion; the means on the 7th day did not differ significantly from those on the control days (P > 0.1, analysis of variance). For this reason the means for all values during aspirin infusion were compared with the preinfusion control values (fig. ). No difference occurred between the control periods and the periods during infusion of aspirin in regard to the slopes of the lines relating acid concentration to net!lux of H + or Na + (P > 0.1, analysis of variance). Although the slopes relating acid concentration to water flux and to PD were 7 H 9 0 :\1 :q ; ~ f ) ~ I J I ) ~ I ) O 00 01J 1)0 1)1J 1K) 1XI 1XI 1J1l In 1J II) lti 1 1J (;-, FI(;. 1. Lesions in antral mucosa near the antral-oxyntic border in a cat that was given 100 mg kg 'day 'of aspirin for 7 days. TAHI.. Summary of {(',..,iuns in intact ('ots that uot an intraven()u... infusion ()f aspirin (or,lialine control) for 7 days :\1). of ('lit..; Incidt'nCl' I.(..;jcln..; :\0, ppr ~ f ( ) l l l a l ' h Sp\'pritytl I 'Icer incidencf:' IIIR k)..! J den 0" R II 1II (J fl/i'. (-):V; /.sot:; 7/11 4n; 7/10 7W:; Il f') I. ~ 1.8 () 1/8 1 : ~ c ; 1/ 17"; 4/11 :{ C ; 1/10 10"; 'J Salirw l'ontrol. h S l ' v f ' incit'x: r i t ~, I null I; I to;) mm ~ ; - ~ ; mm l : ~ extension : through muscularis mucosae.t.

4 November 197 ASPIRIN AND GASTRIC MliCOSAL LSIUNS 757 somewhat flatter during aspmn infusion than during pouch to a solution of 0 mm aspmn in 100 mm Hel control periods, mean responses during aspirin infusion produced the expected large changes in PD and fluxes of were within the range of those obtained during control Ht, Nat, and water (fig. 7). periods. Discussion To show that the methods used could readily detect changes in PD and fluxes that occur when topical barrier This study confirms earlier reports" 10-" that aspirin breakers act, studies were done in 4 other cats with given by extragastric routes can cause lesions of the antral-oxyntic pouches. xposure of the mucosa of the gastric mucosa. The main new findings of this study are: (1) the dose of intravenous aspirin needed to produce lesions is small, with plasma levels within or below the FIG.. Linear lesions at the antral -oxvntic border and puncliform lesions on the crests of oxyntic mucosal f;lds in a cat that was given 50 mg kg - I day I for 7 days. FIG. 4. Ulcer in antral mucosa in the pouch of a 100 mg kg I day I of aspirin for 7 days. ( 'lit FIG. 3. Histological section of 11 chronic ulcer in antral mucosa in a cat that got 5 mg kg 'day ' 0 1' aspirin for 7 days. that was given

5 758 BUGAT TAL. Vol. 71, No..5 Ff(:..'j. Multiple ulcers in the antral mucosa and multiple punctate lesions in the oxyntic mucosa in the pouch of a cat that was given 100 mg kg 'day 'of aspirin for 7 days. ~ -GO o '" " ~ -40 " + r x -0 z > PO,,\CONTROL \ ~ ~,,,, He' mm ~ VOLUM ~.510 " ASPIRIN ~ A, o::--t:.-,lt:.--" ~ o, '- / 'a/control " ~ ~ :;; a, I,, He! mm FIG. b. lectrical putential differen('e PD. net fluxes of H' and Na., and net volume changes in antral-fundic pouches in control testf. and in te:-;ts during ('(mtinuous intravenous infusion of 100 mg kg I day I of aspirin for 7 d a y None ~. of the differences hetween control tpsts and tpsts during aspirin infusion were statisticaljy significant. therapeutic dose range for man, () the lesions include deep ulcers and these occur in antral mucosa near its border wit h oxynt it mucosa, and ( : ~ the ) lesions occur without the changes in PD and ion fluxes that are regarded as signifying that the gastric mucosal barrier to hackdiffusion of H' has been broken. Because low doses of aspirin are effective in producing the lesions, it is unlikely that the toxic effects associated with high doses, such as changes in acid-base balance, 1 playa significant role in their pathogenesis. Oi et at. 17 were the first to emphasize that gastric ulcers in man tend to occur in antral mucosa near its border with oxyntic mucosa. Oi et at. 18 noted that certain kinds of experimental ulcers also show this localization, notably those produced by cinchophen. The ulcers that are seen in man in association with ingestion of large amounts of aspirin also show this same localization. IS Normally hydrogen ions diffuse back into the gastric mucosa at very low rates, so tbat the mucosa is said to have a barrier to such back diffusion. When the mucosa is bathed with certain injurious agents, the rate of back diffusion of H + increases greatly and the barrier is said to be broken. Associated with the increased back diffusion of H+, an increase of entry of Na + into the lumen and a decrease of PD across the mucosa also occur. This triad of increased H + back diffusion, increased N a + entry, and decreased PD is used an an index of the integrity of the gastric mucosal barrier. When these values are normal the barrier is said to be intact and when they are abnormal the barrier is said to be broken. In the present study, aspirin given intravenously did not cause this triad of changes that characterizes the broken barrier. ven when aspirin had caused severe lesions of the gastric mucosa, the triad of changes was not present. This shows that the triad is not a required C :? 0 ~ - 5 0H+ ["1 0 I '"... "0 "0 " :I: ~ X x 400 :::J :::J (1 ii -100 ii z Z Na+ i eoo T PO =t \ /1 C I... '" g w c.!) z <t :I: 3 ~ L U f ~ > u 1''' ,j 3? l T > _ w z I CONTROL, 100 mm HCI 0 mm ASPIRIN t 100 mm HCI 3' 100 mm HCI FIG. 7. gtlel"l of topically applied aspirin solution on el"ctrieal potential difference. PD. and ion and water fluxes in antral fundic pouches. Aspirin b ~ this ' route produced the ex peeled decrease in I'll and changes in net!lux of H'. Na, and water. :\-leans and standard errors for eight tests, two in each of..t cats. 1.

6 November 197 ASPIRIN AND GASTRIC MUCOSAL LSIONS 759 precondition for lesions to form and also that the triad is not a necessary consequence of the presence of lesions. It seems safe to assume that the presence of lesions, particularly those that destroy the full thickness of the mucosa, leads to loss of the barrier function at the site of the lesion. If methods were available to measure the barrier function just at the site of lesions rather than over the whole mucosa, presumably changes would be detectable. Because it is clear from this study as well as from earlier studies o- u that lesions can be present in the stomach without changes in this conventional triad of indices of the integrity of the barrier, it must be concluded that these indices cannot detect localized loss of the barrier caused by lesions even when these involve more than 10% of the area of the mucosa (as was true in the pouches in this study). Because loss of the barrier at the site of lesions cannot be detected by use of this triad of indices, currently there are no means of studying the question of whether this localized loss of barrier precedes the formation of the lesions or is a consequence of the lesions. All of the recognized barrier breakers are effective only by contact with the luminal surface of the mucosa and it must be assumed that they cause alteration in all or most of the mucosa, thus allowing changes to be detected by the conventional triad. There is now strong epidemiological evidence that persons who consume large amounts of aspirin have an increased incidence of gastric ulcer. u Gastric ulcer in persons who do not ingest aspirin is almost always associated with gastritis involving all of the antral mucosa, including the site of the ulcer, and sometimes extending into the oxyntic mucosa. By contrast, gastric ulcers in heavy users of aspirin may occur in stomachs that are devoid of gastritis. It The fact that aspirin users get a form of ulcer not seen otherwise, namely gastric ulcer without gastritis, is further evidence that aspirin causes gastric ulcer. The present study shows that an experimental form of gastric ulcer can be produced by a systemic action of aspirin. Whether a systemic action of aspirin is also involved in human gastric ulcer associated with ingestion of aspirin remains to be determined by future studies. RFRNCS 1. Cooke AR: The role of acid in the pathogenesis of aspirin induced gastrointestinal erosions and hemorrhage. Am J Dig Dis 18:5-3, Davenport HW: Salicylate damage to the gastric mucosal barrier. N ngl J Med , Brodie DA, Chase BJ: Role of gastric acid in aspirin induced gastric irritation of the rat. Gastroenterology 53:04-10, mas S, Swan KG, Jacobson D: Methods of studying gastric secretion. In Handbook of Physiology, Section, Alimentary Canal, vol, Secretion. dited by CF Code. Washington DC, American Physiological Society, 197, p Kontul'1!k SJ: The effect of secretin on gastric acid secretion and peptic ulcers induced by pentagastrin in cats with intact or resected duodenum. Am J Dig Dis 13: , 198. Brodie DA. Tate CL, Hooke KF: Aspirin: intestinal damage in rats. Science Bloom DS, Jacobson D, Grossman MI: Validation of dilution indicators in the stomach. Gastroenterology 5:05-10, Saltzman A: Fluorophotometric method for the estimation of salicylate in blood. J Bioi Chem 174: , Paulus H. Siegel M, Mongan, et al: Variations of serum concentrations and half life of salicylate in patients with rheuma. toid arthritis. Arthritis Rheum 14:57-53, Barbour HG, Dickerson VC: Gastric ulceration produced in rats by oral and subcutaneous aspirin. Arch Int Pharmacodyn Ther 58:78-87, Brodie DA, Hooke KF: ffects of route of administration on the production of gastric hemorrhage in the rat by aspirin and sodium salicylate. Am J Dig Dis 1: , Clark BB, Adams WL: The effect of acetylsalicylic acid on gastric secretion. Gastroenterology 9:41-45, Cooper GN, Meade RC, llison H: Heidenhain pouch bleeding due to oral salicylates. Arch Surg , Gottschalk A, Menguy R: Role of gastric acid in aspirin-induced erosive gastritis. Proc Soc xp Bioi Med 135: , Lynch A, Shaw H, Milton GW: ffect of aspirin on gastric secretion. Gut 5:30-3, ichenholz A, Mulhausen RO, Redleaf PS: Nature of acid base disturbance in salicylate intoxication. Metabolism , Oi M, Oshida K, Sugimura S: The location of gastric ulcers. Gastroenterology 3: Oi M, Toriumi T, Miho 0, et al: Location of experimental ulcers as compared with that of human peptic ulcer. In Peptic Ulcer. dited by CJ Pfeiffer. Copenhagen, Munksgaard, 1971, p MacDonald WC: Correlation of mucosal histology and aspirin intake in chronic gastric ulcer. Gastroenterology 5: , Cooke AR, Kienzle MG, Tubbesing TJ: ffect of an ulcer on the gastric mucosal barrier in dogs. Digestion 11: Moody FG, Aldrete JS: Hydrogen permeability of canine gastric secretory epithelium during formation of acute superficial ero. sions. Surgery , Gerety DC, Guth PH: Restraint induced gastric erosions. Role of acid back diffusion. Am J Dig Dis , Levy M: Aspirin use in patients with major upper gastrointestinal bleeding and peptic ulcer disease. N ngl J Med , Cameron AJ: Aspirin and gastric ulcer. Mayo Clin Proc 50:55-570, Chapman BL, Duggan JM: Aspirin and uncomplicated peptic ulcer. Gut 10: , 199