MECHANISM OF STRESS ULCER

Transcription

1 GASTROENTEROLOGY 66: , 1974 Copyright 1974 by The Williams & Wilkins Co. Vol. 66, No.4 Printed in U.S.A. MECHANISM OF STRESS ULCER II. Differences between the antrum, corpus, and fundus with respect to the effects of complete ischemia on gastric mucosal energy metabolism RENE MENGUY, M.D., AND Y. F. MASTERS Department of Surgery, University of Rochester School of Medicine and Dentistry and The Genesee Hospital, Rochester, New York Recently we proposed that the event linking hemorrhagic shockinduced gastric mucosal ischemia and "stress ulceration" may be a rapid and profound decrease in gastric mucosal energy metabolism. A characteristic feature of stress ulcers caused by hemorrhage, both in man and in experimental animals, is a tendency to involve only the corpus and fundus and spare the antrum. Therefore, if our hypothesis is to remain valid, one would have to show that the antrum is better able to sustain its energy metabolism in the face of ischemia. The data on which this report is based indeed indicate that, under conditions of complete ischemia, dephosphorylation of the adenylate pool is significantly less marked in the antrum than in the corpus and fundus. The nature of the event or events linking shock-induced gastric mucosal ischemia and the development of acute gastric mucosal erosions remains unknown. Recently, we have proposed an explanation for acute gastric mucosal erosions developing in rats subjected to hemorrhagic shock. 1 We found that nonlethal shock induced in rats by withdrawing a volume of blood equivalent to 2.0% of the animal's weight caused, within 15 min, a 75% reduction in adenosine triphosphate (ATP) levels in gastric mucosal tissue, a more severe reduction than was observed in hepatic or skeletal muscle tissue from the same animals. In another series of experiments, we found that when these tissues were subjected to complete, warm ischemia, a deficit in energy metabolism occurred more rapidly in Received August 9, Accepted October 30, 1973, Address requests for reprints to: Dr. Rene Menguy, Surgeon-in-Chief, The Genesee Hospital, 224 Alexander Street, Rochester, New York This work was supported by Grant AM from the United States Public Health Service and by the George A, Bernard Surgical Research Fund. 509 gastric mucosa than in liver and more rapidly in liver than ill muscle. 1 On the basis of these data, we proposed that a profound decrease in energy metabolism in the gastric mucosa is the principal cause of the cellular necrosis occurring within minutes after the induction of hemorrhagic shock. An interesting peculiarity of "stress ulcers," particularly those precipitated by trauma or hemorrhage, has offered us an opportunity to either invalidate or support our hypothesis. This is the tendency of the lesions to involve only the more proximal regions of the gastric mucosal surface. This feature has been recognized in human patients,2 and has been described more accurately in animals subjected to hemorrhagic shock: the rabbit,3. 4 the pig,5 and the dog. 6 In our work with bled rats, in which gross gastric mucosal lesions are invariably obvious within 45 to 60 min after the induction of shock, we have found that the erosions occur in the parietal cell area and never in the antrum. If our hypothesis is valid, one should be able to demonstrate that energy metabolism in the

2 510 MENGUY AND MASTERS Vol. 66, No.4 antral mucosa is less vulnerable than that of the fundus to ischemia. In other words, the finding in the antrum of an energy deficit greater or equal to that occurring in the fundic mucosa under identical ischemic conditions, would invalidate the energy deficit hypothesis as applied to the explanation of stress ulceration. The converse would, if not prove our hypothesis, at least support it. The data presented in this paper will show that in the stomachs of rats and rabbits rendered completely ischemic, the characteristic deficit in energy metabolism caused by ischemia is significantly less severe in the mucosa of the antrum than in that of the corpus or fundus. Materials and Methods Rats and rabbits were used for this experiment. Rats. Male Holtzman rats weighing approximately 300 g were used. On the evening before the experiment, food was removed and drinking water was replaced with 5% solution of sucrose containing a salt mixture. At 8 AM the following morning, the nutrient solution was removed, and the experiment was begun at 10 AM. Laparotomy was done under urethane (125 mgper 100 g) anesthesia. A ligature was placed around the esophagus immediately above the stomach with care not to occlude the major gastric vascular pedicle. At the appropriate moment, the stomach was lifted from its bed by slight traction on the ligature, and, with two quick scissor strokes, was severed from its connections. It was left in situ for 30, 60, or 120 sec and then stop-frozen by immersion in liquid N 2. Controls were obtained by immersing in liquid N 2 stomachs which had been removed in the same manner and under the same conditions, but without an ischemic interval. Control or test experiments were paired in random order. Frozen tissues were lyophilized. The dry tissue was prepared for biochemical analysis in the following fashion. Food debris and hair were carefully removed by gentle suction from the gastric mucosal surface. The glandular stomach was divided into two portions: corpus and antrum, division for which landmarks are quite obvious in the rat stomach. For additional processing, and to obtain sufficient antral tissue for biochemical analysis, two antra and two corpora, respectively, from a paired experiment were pooled. Each pool represented one antral, or corpus, tissue sample for analytical and statistical purposes. Mucosa was scraped off the underlying muscularis mucosae. Mucosal scrapings were pulverized in a mortar, and the dry tissue powder was then subjected to biochemical analysis. Rabbits. Male albino rabbits weighing approximately 2000 g were used. On the evening before the experiment, food was removed and drinking water was replaced with a 5% solution of sucrose to which a salt mixture was added. The nutrient solution was removed at 8 AM the following morning, and the experiment was started at 10 AM. Laparotomy was done under Nembutal (40 mg per kg) anesthesia. A ligature was placed around the esophagus. At a given moment (always the same with respect to the induction of anesthesia), the stomach was lifted from its bed by traction on the ligature and quickly severed from its connections. The stomach was left in situ for 60, 120, or 180 sec and then immersed in liquid N 2. For the harvesting of control gastric tissues, the procedure was the same up to the step when the stomach was momentarily lifted from its bed. At this point, the entire stomach was crushed between two large aluminum paddles precooled to the temperature of liquid N 2. Portions of tissue extruding beyond the paddles were severed and the entire sample was dropped into liquid N 2. Frozen stomachs were trimmed of mesenteric fat and then lyophilized. Dry tissues were opened, and samples of antrum, corpus, and fundus were taken, as described in figure 1. Every effort was made to maintain consistency in the cutting of the specimen by using the serosal vascular pattern as landmarks and by having the same individual perform this step throughout the experiment. Cleansing of the mucosal surface, scraping the mucosa off the muscularis mucosae, and pulverizing it prior to biochemical analysis was identical to the procedures used for the rat stomachs. Tissue powders were extracted for 10 min in 0.5 Mice-cold perchloric acid in all glass Potter, Elvehjem homogenizers. After centrifugation the supernatants were neutralized with 2.0 M KHCO a, and estimations of ATP, adenosine diphosphate (ADP), adenosine monophosphate (AMP) and free glucose were carried out. Estimations of glucose-6-phosphate, pyruvate, and lactate were done, in addition to the latter, in rabbit mucosa. ATP tissue levels were determined with phosphoglycerate kinase according to Adam.' ADP and AMP were estimated with lactic dehydrogenase, pyruvate kinase, and myokinase according to Adam.' At regular intervals, replicate analyses of fresh, pooled gastric mucosa were carried out with these methods. The highest coefficients of variation

3 April 1974 MECHANISM OF STRESS ULCER. II 511 measured were 3.2,2.4, and 6.6% for ATP, ADP, and AMP, respectively. Also, analytical recovery of ATP from freshly prepared standard solutions containing low and high concentrations of ATP was measured from time to time. The "worst" recoveries were 106% ± 0.8% (SE) and 97% ± 0.7% (SEj for low and high concentrations of ATP, respectively. Pyruvate and lactate were estimated with lactic dehydrogenase. 7 Free glucose was determined with hexokinase and FIG. 1. Schematic illustration of the portions of the rabbit stomach which were sampled for biochemical analysis. glucose-6-phosphate dehydrogenase according to Slein. 7 Glucose-6-phosphate was determined with glucose-6-phosphate dehydrogenase according to Bergmeyer.7 Glycogen concentration (rabbit mucosa only) was estimated according to Hassid and Abraham. s Substrate concentrations were expressed as micromoles per gram of tissue (dry weight). Glycogen concentrations were expressed as micromoles of glycogenglucose per g of tissue (dry weight). Results Rats. Control data for the experiments with rats are given in table 1. Levels of ATP as well as those of (ATP + ADP + AMP) were significantly higher in the mucosa of the corpus than in the antrum. Changes in the levels of these substrates during complete ischemia are summarized in table 2. As we had previously reported, complete ischemia caused an immediate dephosphorylation of adenine nucleotides. The rate of dephosphorylation was significantly greater in the mucosa of the corpus than in antral mucosa as shown by the following: in corpus mucosa (1) ATP levels decreased at a greater rate than in the antrum; (2) AMP levels increased at a greater rate than in the antrum; (3) ADP levels were lower than in the antrum; (4) the energy charge of the adenylate pool [(ATP) +O.05(ADP) ]/[(ATP) +(ADP) + (AMP)], a parameter which provides a quantitative estimate of the energy state of the cell,9 dropped more rapidly than in the antrum. (This value, which can be obtained from a set of' concentrations of ATP, ADP, and AMP, expresses the ex- TABLE 1. Substrate leuels in the fundic and antral mucosa of nonischemic rat stomachs a Antral mucosa (8)" Fundic mucosa (8)" ATP" ± ± 0.76 ADp d ± ± 0.28 AMP"..., ± ± 0.04 ATP+ADP+AMP ± ± 0.77 Energy charge' ± ± 0.01 Glucose ± ± 0.56 P <0.001 <0.01 NS' <0.001 NS <0.01 avalues equal micromoles of substrate per gram of tissue (dry weight) ± SD. h Numbers in parentheses indicate numbers of pairs of tissue samples pooled and studied., p, significance of difjerences between antrum and fundus. " ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate., NS, not significant. 'Adenylate pool enen;y charge [(ATP)+0.5(ADP)]/[(ATP)+(ADP)+(AMP)].

4 TABLE 2. Influence of complete gastric ischemia on substrate levels in the fundic and antral mucosa of the rat stomach.' h 30 sec 60 sec 120 sec Antral mucosa Fundic mucosa p Antral mucosa Fundic mucosa p Antral mucosa Fundic mucosa p (7) (7) (7) (7) (6) (6) ATP" ± 8%" - 38 ± 7%" < ± 9%" -56 ± 9%d < ± 6%d -64 ± 6%" < 0.01 ADP" ± 27%" +55 ± 20" NS' +109 ± 34%" +61 ± 22%" < ± 19%d + 17 ± 5%d < 0.01 AMP" ± 33%" +179 ± 28%" < ± 72% +395 ± 51% < ± 85%d +680 ± 58%" < ATP+ ADP+ AMP. -8 ± 6%' - 15 ± 6%" NS - 1 ± 5% (NS) - 18 ± 8%d < ± 7%' - 20 ± 5%d < 0.05 Energy charge' ± 3%" -18 ± 2%" < ± 5%d -33 ± 5%' < ± 5%" -46 ± 4%" < Glucose ± 15%' - 33 ± 8%" < ± 18%(NS)i - 22 ± 5%d NS a Values equal average percentage of change from mean control values given in table 1. P indicates significance of differences between antrum and fundus. h 30 sec, 60 sec, 120 sec indicate the intervals between interruption of blood supply and application of "stop-freeze" to tissue. Numbers in parentheses indicate pairs of tissue samples pooled and studied. C ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate. d Significance of percentage of change from mean control value, P < ens, not significant. I Significance of percentage of change from mean control value, 0.01 < P < 0.05., Significance of percentage of change from mean control value, < P < Adenylate pool energy charge [(ATP) +0.5(ADP))/[(ATP) +(ADP)+(AMP)]. Cll I-' c:: "< ).. rn rn ""

5 April 1974 MECHANISM OF STRESS ULCER. II 513 TABLE 3. Substrate levels in the mucosa of the antrum, corpus, and fundus of nonischemic rabbit stomachs a Antrum (6)' Corpus (6)" Fundus (6)' P" P" P" ATP" ± ± ± 1.28 <0.001 NS' <0.001 ADP" ± ± ± 0.25 <0.001 <0.001 NS AMP" ± ± ± 0.17 <0.05 NS NS ATP+ADP+AMP ± ± ± 1.35 <0.001 NS <0.01 Energy charge' ± ± ± 0.01 NS <0.02 <0.02 Pyruvate ± ± ± 0.06 NS NS NS Lactate ± ± ± 0.42 NS <0.05 <0.05 Lactate/pyruvate...,.,, ' 6.14 ± ± ± 1.03 NS NS NS Glucose ± ± ± 2.07 NS NS NS Glucose-6-P ± ± ± 0.09 <0.02 <0.001 NS Glycogen ± ± 3.21 NS a Values equal micromoles of substrate per gram of tissue (dry weightl ± SD. 'Numbers in parentheses indicate numbers of separate tissue samples studies. C pi, antrum versus corpus; p2, antrum versus fundus; p3, corpus versus fundus. d ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate. ens, not significant. t Adenylate pool energy charge [(ATP)+0.5(ADPl)/[(ATPl+(ADP)+(AMP)). tent to which the adenine phosphate system is saturated with high energy phosphate bonds. When all of this adenosine system is in the form of ATP, it is completely saturated and has a theoretical energy charge of 1.0. Were all of the adenine nucleotides to be in the form of AMP, the system would be devoid of high energy bonds and its energy charge would be 0.0.) Rabbits. Control data for the experiments with rabbits are given in table 3. As was noted in the rat stomach, levels of ATP as well as those of (ATP+ADP+AMP) were significantly higher in the corpus mucosa than in that of the antrum. ADP and AMP levels were significantly lower in the antral mucosa than in that of the corpus. In the fundus, ATP levels were lower than in the corpus. The other notable difference among these three regions of the nonischemic gastric mucosa was a significantly lower level of glucose-6-phosphate in the antral mucosa than in that of the corpus or fundus. Changes in the levels of the substrates studied during complete ischemia are given in tables 4 and 5 and figure 2. In the mucosa of the corpus and the fundus: (1) ATP levels dropped at a faster rate than in the antrum; (2) AMP levels increased at a faster rate than in the antrum; (3) ADP levels, after 60 sec, were lower than in the antrum; (4) the energy charge of the adenylate pool decreased more rapidly than in the antrum. These findings indicate that in rabbits, as in rats, complete gastric ischemia caused a more rapid dephosphorylation of adenine nucleotides in corpus and fundus than in antrum. Although the energy deficit, as measured by the parameters enumerated above, was slightly more severe in the fundus than in the corpus, this difference was not significant. The difference between the antrum, on one hand, and the corpus and fundus, on the other, was also striking with respect to increases in levels of glucose-6-phosphate which were approximately 10 times greater in antral mucosa. Glycogen levels (not estimated in fundic mucosa), began to drop immediately in the antrum, whereas in the corpus a significant drop in glycogen levels did not occur until after 120 sec of ischemia. With respect to the other substrates estimated (lactate, pyruvate, and free glucose), differences among the three regions of the gastric mucosa were not significant. Discussion In all animal models of shock-induced stress ulceration studied to date, the mucosal lesions spare the antrum. On the basis

6 TABLE 4. Influence of complete ischemia on substrate levels in the mucosa of the antrum, corpus. and fundus of the rabbit stomach a. 60 sec 120 sec 180 sec Antrum (6) Corpus (6) Fundus (6) Antrum (6) Corpus (6) Fundus (6) Antrum (6) Corpus (6) Fundus (6) ATP' ± 13%" -54 ± 4%' - 57 ± 9%' - 46 ± 10%' - 61 ± 7%' - 65 ± 3%' - 43 ± 8%' -67 ± 3%'! - 66 ± 3%' ADP' ± 32%' +96 ± 13%' + 70 ± 16%' + 82 ± 27%' + 47 ± 10%' +41 ± 40% ± 8%' + 52 ± 19%" t +41 ± 27%d AMP' ± 64%d ± 43%' +704 ± 86%' +283 ± 42%' ± 1\2%' ± 114%' +285 ± 72%' t ± 89%' +830 ± 68%' ATP+ ADP+AMP -8 ± 14%' -7 ± 50/0' No change - 16 ± 9%" - 8 ± 8%' - 5 ± 14%1-12 ± 6%1-12 ± 3%" - 7 ± 2%' Energy charge h -15 ± 4%' - 37 ± %' 2-43 ± 5%' - 26 ± 5%' -47 ± 4%' - 51 ± 2%' - 24 ± 3%' -51 ± 3%' - 51 ± 2%' Pyruvate ± 23%' -15 ± 29%' - 15 ± 9 %' - 36 ± 14%' - 36 ± 13%d + 6 ± 30'JIoI - 33 ± 11%' - 33 ± 33%1-1 ± 26%1 Lactate ± 29%' +7 ± 29%' +19 ± 20%' +24 ± 55%' +16 ± 36';" ± 31%" + 28± 41%' + 9 ± 31%1 +54 ± 27%" Lactate/pyruvate ± 36%' +55 ± 90%' +38 ± 23%" ± 95%" +84 ± 41%d +41 ± 41% ± 40%d + 99 ± 90%" +-59 ± 36%d Glucose ± 18%' -17 ± 14%' + 8 ± 14%' + 20 ± 37%' -14 ± 13 9' ± 12%' - 6 ± 11 %1-27 ± 12 % %' Glucose-6-P ± 50%' +14 ± 32%' +48 ± 45%" ± 158%' + 49 ± 34%' +59 ± 33%d ± 133%' + 21 ± 14%' + 22 ± 40%' Glycogen ± 21%" + 51 ± 32%" - 39 ± 22%" - 6 ± 30o/r/ - 6 ± 12%1-49 ± 8%' - 2 ± 32%1 I a Values equal average percentage of change from mean control values given in table 3. h 60 sec, 120 sec, 180 sec indicate the intervals between interruption of blood supply and application of "stop-freeze" to tissue. Numbers in parentheses indicate numbers of separate tissue samples studies., ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate. d Significance of percentage of change from mean control values, < P < 0.01., Significance of percentage of change from mean control values, P < I NS, not significant. " Significance of percentage of change from mean control values, 0.01 < P < h Adenylate pool energy charge [(ATP) + 0.5(ADP)]/[(ATP) + (ADP)+(AMP)]. CJl... G:l c::: -< :» V) V).gs ""

7 April 1974 MECHANISM OF STRESS ULCER. II 515 TABLE 5. Influence of complete ischemia on substrate leuels in the mucosa of the antrum, corpus, and fundus of the rabbit stomach (significance of the differences among the three tissues with respect to average percentage of changes from control ualues)" 60 sec 120 sec 180 sec P' P' P' P' P' P' P' P' P' ATP' <0.01 <0.001 NS' <0.02 <0.01 NS <0.001 <0.001 NS ADP' NS NS <0.02 <0.02 NS NS <0.01 <0.01 NS AMP' <0.001 <0.001 <0.01 <0.001 <0.001 NS <0.001 <0.001 NS ATP+ADP+AMP. NS NS NS NS NS NS NS NS <0.01 Energy charge" <0.001 <0.001 <0.01 <0.001 <0.001 NS <0.001 <0.001 NS Pyruvate NS NS NS NS <0.05 <0.02 NS <0.02 NS Lactate NS NS NS NS NS NS NS NS <0.05 Lactate/pyruvate. NS NS NS NS NS NS NS NS NS Glucose.. NS NS <0.02 NS NS <0.01 <0.01 NS <0.02 Glucose-6-P.. <0.001 <0.001 NS <0.001 <0.001 NS <0.001 <0.001 NS Glycogen <0.001 NS NS a 60 sec, 120 sec, 180 sec indicate the intervals between interruption of blood supply and application of "stop-freeze" to tissue. P', antrum versus corpus; p2, antrum versus fundus; P 3, corpus versus fundus., ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate., NS, not significant. "Adenylate pool energy charge [(ATP)+0.5(ADP)J/[(ATP)+(ADP)+(AMP)). 8 q CHARGE I (A.OOI --/8 'a '.! A', p<.001 p<.ooi C ' "'-...1 '=-,. '. 1"i /_A., p<.ooi " _! -''' p.001 \ P-T I p<.ooi \,.6 '----T-- c.4.z:.:jc 1,---6,.1,O-""12\o."O""''''''1'*'80''' 2! -> " >- 0+.". -'" +> - >."." - oo SECONDS AMP,c Ai-"- ---f // p<.ooi A, p<.ooi (!r;-! FIG. 2. Influence of complete ischemia of , and 180 sec duration on adenine nucleotide levels in the mucosa of the antrum and corpus of the rabbit stomach. For adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) each point on the graphs represents the mean (of six experiments) percentage of [(ATP)+(ADP)+(AMP)] at 60,120. and 180 sec after the complete interruption of gastric blood flow. Note that this value is the same for all three variables in nonischemic tissues. Charge energy charge of the adenylate pool. Each point represents the mean (of six test experiments at each time period or six control-zero-time-experiments) absolute value of this parameter. Note that the energy charge is identical in nonischemic antral and corpus mucosa. A, antral mucosa; C, corpus mucosa; values of P refer to the significance of differences between the antrum and corpus in relation to their respectve changes from control values at varying periods of ischemia. To simplify the graph. data from fundus which were similar to those from corpus, are omitted.

8 516 MENGUY AND MASTERS Vol. 66, No.4 of previous studies, we have proposed that shock-induced mucosal injury results from a severe gastric mucosal energy deficit. 1 If this hypothesis were incorrect, any energy deficit developing in antral mucosa during ischemia would be as great as, or greater than, that developing in parietal cell mucosa. The findings presented in this report, findings which indicate that the converse occurs, do not allow us to reject the hypothesis; nor should they be interpreted in any way as a verification of the hypothesis, since it is quite possible that shock may injure the gastric mucosa via some mechanism other than an energy deficit. The reason for studying energy metabolism in antral and parietal cell mucosa from completely ischemic stomachs, rather than from stomachs of shocked animals, is that, under the latter conditions, any measured differences between these two tissues could be ascribed to selective regional ischemia. In studies now being prepared for publication, we have found that breakdown of high energy phosphates in the gastric mucosa of shocked rabbits is also considerably more rapid in the parietal cell mucosa than in the antrum. From the data presented herein, we can assume that these differences do not result from selective, regional ischemia in the gastric mucosa of rabbits during shock. We cannot explain the greater tolerance of antral mucosa to ischemia. However, the data do allow us to reject the possibility that the slowly developing energy deficit in antral mucosa is associated with a more active anaerobic energy production. The increase in glucose-6-phosphate in ischemic antral mucosa indicates that this intermediate is accumulating, probably due to inhibition of the phosphofructokinase reaction. Thus the slowly developing energy deficit in ischemic antral mucosa is associated with a slow glycolytic flux. It is possible that the different tolerances of antral and parietal cell mucosa to ischemia result from different energy demands existing at the time the tissues become ischemic. REFERENCES 1. Menguy R, Desbaillets L, Masters YF: Mechanism of stress ulcer: influence of hypovolemic shock on energy metabolism in the gastric mucosa. Gastroenterology 66:46-55, Lucas C, Sugawa C, Walt A: Natural history and surgical dilemma of "stress" gastric bleeding. Arch Surg 102: , Harjola PT, Sivula A: Gastric ulceration following experimentally induced hypoxia and hemorrhagic shock. Ann Surg 163:21-28, Skillman JJ, Gould SA, Chung RSK, et al: The gastric mucosal barrier: clinical and experimental studies in critically ill and normal man, and in the rabbit. Ann Surg 172: , Goodman AA, Osborne MP: An experimental model and clinical definition of stress ulceration. Surg Gynecol Obstet 134: , Safaie-Shirazi S, DenBesten L, Hamza KN: The role of vagotomy in preventing bile salt-induced stress ulcers during hemorrhagic shock. J Trauma 12: , Bergmeyer HU (editor): Methods of Enzymatic Analysis. New York and London, Academic Press, Hassid WZ, Abraham S: Methods in Enzymology, vol 3. Edited by SP Colowick, NO Kaplan. New York, Academic Press, 1957, p Atkinson DE: The energy charge of the adenyl ate pool as a regulatory parameter. Interaction with feedback modifiers. Biochem J 7: , 1968