- Email: [email protected]
Effect of cimetidine on canine gastric mucosal pH and blood flow
Effect of Cimetidine on Canine Gastric Mucosal pH And Blood Flow
Laurence Y. Cheung, MD, St. Louis, Missouri Leonard A. Sonnenschein, BS, St. Louis, Missouri
Cimetidine, an Hz-receptor antagonist, is widely used to inhibit acid secretion in patients with chronic duodenal or gastric ulcers [I]. However, its efficacy in the prevention and treatment of acute upper gastrointestinal bleeding in severely ill patients is uncertain [2-71. Since gastric blood flow [8,9] and the intramural pH of the gastric mucosa [IO--131 play an essential role in mucosal tolerance to acute ulcerogenesis, it is desirable to know the effects of cimetidine on the gastric microcirculation and intramural PH. Konturek et al [14] demonstrated in dogs that metiamide inhibition of acid secretion stimulated by several secretogogues was associated with a significant reduction in mucosal blood flow as measured by aminopyrine clearance. Delaney et al [15] reported that cimetidine caused no change in total gastric and mucosal blood flow as determined by radioactive microspheres to any portion of the stomach in unstimulated dogs. The discrepancy between these two reports has been attributed to variables in the methods of measuring mucosal blood flow and to the different secretory states of the canine stomach when H2-receptor antagonists were administered [15]. Therefore, this study was designed to compare the effect of cimetidine on mucosal blood flow as determined by microspheres with that measured by carbon-14 aminopyrine clearance. In addition, the effect of secretory inhibition by cimetidine on the intramural pH of the gastric mucosa was also evaluated. From the Surgical Service of the Veterans Administration Hospital, and the Department of Surgery, Washington University School of Medicine, St. Louis, Missouri. This investigation was supported by the Medical Research Service of the Veterans Administration and by grant AM25998-03 from the National institute of Health, Bethesda, Maryland. Requests for reprints should be addressed to Laurence Y. Cheung, MO, Department of Surgery, Washington University School of Medicine, 4960 Audubon Avenue, St. Louis, Missouri 63110. Presented at the 23rd Annual Meeting of the Society for Surgery of the Alimentary Tract, Chicago, Illinois, May 18-19, 1982.
24
Material
and Methods
Mongrel dogs that weighed from 20 to 25 kg were anesthetized with sodium pentobarbitol (25 mg/kg) and maintained on a Harvard respirator throughout each experiment. Polyvinyl catheters (inside diameter (ID) 0.058 inch) were placed in both femoral veins for infusion of fluid, blood, and drugs. Arterial pressure was monitored through a catheter inserted into the left femoral artery (Statham transducer P23AA; Hewlett Packard direct writing recorder). A catheter (ID 0.067 inch) was placed in the left ventricle through the right carotid artery. A chambered segment of gastric corpus with an isolated vascular pedicle was prepared as described by Moody and Durbin [16]. This chamber provided a mucosal surface of approximately 36 cm2. In all experiments, isotonic hydrogen chloride solution (0.15N) was placed into and recovered from the chamber at 15 minute intervals. The secretory volume was calculated as the change in weight between the instilled and recovered solutions. Hydrogen ion flux was determined by potentiometric titration of instillate and recovered samples to a pH of 6.5 (Radiometer, Copenhagen) with O.lN sodium hydroxide. Venous flow through the gastric segment was measured by occluding the gastrosplenic vein proximal to a Silastic@ catheter (ID 0.56 inch) placed in the superior splenic vein. Venous pressure was continuously monitored by a polyvinyl catheter (ID 0.058 inch) placed in a terminal splenic vein (Statham transducer P23BB). For each 15 minute period, timed venous samples were collected at baseline pressure in preweighed tubes containing EDTA. Volume was calculated on the basis of 1 g/ml. Details of the technique of measuring total gastric and mucosal blood flow with y-labeled microspheres have been reported previously by Archibald et al [17]. In brief, three measurements were made in each dog by injecting microspheres labeled with strontium-85, cerium-141, or chromium-51. The first injection was given during a rest period. The second and third injections were given during the steady state period of histamine-stimulated acid secretion and cimetidine inhibition. For each injection, approximately 100,000 to 500,000 spheres of 15 f 5 ~1in diameter were injected from a mixing chamber into the left ventricle.
The American
Journal of Surgery
Cimetidine and Gastric Blood Flow
A reference sample was drawn at 10 ml/min from the distal aorta for 80 seconds, beginning 10 seconds before injection. The amount of blood loss from arterial and venous sampling was replaced by transfusion. After the completion of each experiment, the gastric mucosa, submucosa, and muscularis were bluntly separated and dried. The tissue specimens were dissolved in 15 percent ethanolic potassium hydroxide solution, centrifuged, and then counted in a gamma counter. Flow was calculated by comparing the activity of each isotope in the tissue with that in the reference sample. Carbon-14 aminopyrine clearance was measured as described by Tague and Jacobson [18]. Carbon-14 aminopyrine (Amersham-Searle, 52.8 @Zi/mg) in a loading dose of 0.15 &i/kg was injected intravenously over 2 minutes. This was followed by continuous intravenous infusion of carbon-14 aminopyrine (0.08 &i/kg per hour). Each animal was equilibrated with aminopyrine for 1 hour before starting the study. At hourly intervals, 10 ml blood samples were drawn into heparinized tubes from the femoral arterial catheter and centrifuged at 4’C to obtain plasma. Mucosal bathing solution was instilled and recovered at 15 minute intervals. One ml of gastric bathing solution was pipetted directly into a scintillation vial and mixed with 10 ml of phase combining system (Amersham-Searle). Plasma samples were extracted as described by Tague and Jacobson [18] and mixed with 15 ml of phase combining system for cell counting. Appropriate blanks were also counted. Gastric and plasma samples were counted for 10 minutes. After subtraction of blanks, cpm/ml was determined and the following equation used to calculate clearance: clearance ml/min = cpm/ml gastric juice/cpm/ml plasma X ml/min gastric juice secreted plus bathing fluid. The intramural pH was measured using a miniature pH electrode (Microelectrode Inc., Londonderry, NH) as decribed by Kivilaakso et al [10,11]. An area of 0.5 to 1.0 cm2 of the gastric wall was denuded of its seromuscular coat. The electrode was directly advanced from the serosal surface through the denuded area into the underlying mucosa at a micrometer depth of 500 p using a micromanipulator (Narishige, Tokyo). The denuded area was
coated with a drop of sesame oil to prevent excessive evaporation and carbon dioxide loss. A calomel reference electrode was connected to the serosal surface of the chamberthrough a saline-agarbridge.The millivoltagewas recorded by a high input impedance pH electrometer (Radiometer).Measurementsof intramuralpH were made continuously throughout each experiment. The average millivoltage was converted to pH units using a calibration curve. The microelectrodes were calibrated in a series of nonphosphate buffers at 37V. The pH response curve was strictly linear, at least over a pH range of 5 to 9. The approximate location of the microelectrode in the gastric wall during the pH measurements was studied at the end of experiments in several dogs by generating a lesion around the tip of the electrode by electrocoagulation. The tissue damage was identified by a pathologist with light microscopy. All of the identified lesions were situated in the deep third layer of the mucosa near the muscularis mucosa. After three resting periods, histamine was infused intravenously at 1.0 pg/kg per minute for the remaining 14 periods. The acid secretion usually reached a plateau 4 periods after the beginning of histamine infusion (period 7) and remained at a steady state throughout the experi-
Volume 145,
January 1983
IV Ctmetldme IV Hlstomtne
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Figure 1. Sequenttal changes in secretory votims and total ackt output In six expertlments, expressed as ths maan f standard enorofths-HkstanVne wask&ssdtntravenousQat ipg/kg per minute. Cknatkikts was given tntravenousty (50 pglkg per mbnde) In addttb to the background htstamhm.
ment. Cimetidine (50 pg/kg per minute) was added intravenously to the background histamine infusion from periods 10 through 13. The dose chosen for cimetidine was the minimum that would consistently give nearly 100 percent inhibition of histamine-stimulated acid secretion. The results from each period were pooled and represented by mean plus or minus the standard error of the mean. The data was analyzed with the paired Student’s t test.
Results The sequential changes in the acid secretion and secretory volume are shown in Figure 1. Before histamine infusion, the secretory volume was negligible. In fact, there was a small net hydrogen ion loss from the lumen as calculated by the change in the amount of hydrogen ion between the instilled and recovered solutions. Intravenous infusion of histamine stimulated the total acid secretion as well as the volume (p
Cheung and Sonnenschein
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PERIODS (I5 MINI F&we 3. Seqnwtlal changes h cahm-14 amhqyrbe chwrame and mucosal blood flow (n = 8) as detemdnedby mkroepheres
PERIODS (I5 MINI [email protected]~hl-nowandtotalgastrkbhmd flowmeasomdbymkmqhms &lfngpl9rws3,9,aml13hflw experhents, expressed as the mean f standard error of the
mean. Htstamh waskhsedhtrawmouslyat 1pgikgwmhte. Clmetidhe was g&n intravenous/y(SO pg/kg per mhte) addMon to backgiwnd hlatamkte.
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trated. Intravenous infusion of histamine resulted in a significant increase in total venous flow (p <0.05) and total gastric blood flow as determined by microspheres (p <0.02). Cimetidine inhibition of acid secretion was accompanied by a reduction in total gastric blood flow as measured by venous flow (p <0.02) and by microspheres (p <0.05). Paired t test analysis showed no significant difference between the total gastric blood flow of cimetidine-inhibited periods and that of the prestimulated periods. Total gastric blood flow measured simultaneously by venous outflow correlated well with microsphere-determined blood flow. The linear regression revealed a highly significant correlation (r = 0.91, slope = 0.97, n = 15, p
26
duringperiods 3, g, and 12, expressedas the mean f standard emwofthemean. Hlstamhe washhsedhtraveaus&at 1Wkg per minute. Cknettdhe was given Intravenously( 50 pgf kg per m/note) In addtth to backgrwmd hlstamlne.
(p <0.02). Despite the background histamine infusion, cimetidine inhibition of acid secretion was accompanied by a significant reduction in carbon-14 aminopyrine clearance and microsphere-determined mucosal blood flow. Mucosal blood flow during the cimetidine inhibition period (period 13) was not significantly different from that in the prestinudated period (period 3). In Figure 4 the percent increase in blood flow during histamine infusion and the percent reduction in blood flow after cimetidine inhibition of acid secretion are shown. Histamine infusion resulted in a significant increase in total blood flow as measured by venous flow and microspheres and mucosal blood flow as indicated by microspheres and carbon-14 aminopyrine clearance (p cO.05). The degree of increase in mucosal blood flow was slightly, but not significantly, higher than total gastric blood flow. Cimetidine inhibition resulted in a significant reduction (p <0.05) in total gastric blood flow and mucosal blood flow as measured by all these methods. There was no significant difference between cimetidine-inhibited periods and prestimulated periods in any measurement, which indicated that total and
The American Journal of Surgery
Cimetidine and Gastric Blood Flow
I.V. Cimetidlne
I.\/: Histamine
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0 IA 0 Flgore 4. Percent changesIn blood flow duringhktamlne sflmuIatkn and clmetldlne Inhlbltlonof add secretton.All data were expressed as the mean l standard error of the mean of five experlments except canbon-14 amlnopyrlneclearance ( n = 8).
mucosal blood flow returned to the preinfusion level when secretion was inhibited by cimetidine. In Figure 5 the sequential changes of the intramural pH of gastric mucosa during histamine stimulation and cimetidine inhibition are shown. In control dogs, the intramural pH of the gastric mucosa increased significantly (p
Valume 145, January 1333
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PERIODS (I5 MINI Figure 5. Effects of intravenoushfstamfneand clmetldlne on the lntramwralpH of the gastrk nucosa. ln conttvl &gs, intravenous hlstamlne slgnlfkantly increased the intramuralpH ( p < 0.00 1) when compared with psrkds before hlstamlneInfusion.llm addMonof clmsthilneto backgmundh&am&m sWfkant& reduced the Intramural pH (p <0.007) when compared wlth hlstamlne lnfuslonakne.
drochloric acid requires the utilization of energy and oxygen. Since such a metabolic demand is no longer present during cimetidine inhibition [20], one would expect that gastric blood flow would return to the resting level. The effect of a secretory inhibitor on gastric microcirculation may vary depending on whether it is administered during resting or during stimulated conditions [21]. In the study reported by Delaney et al [15] cimetidine was administered in anesthetized dogs without the background histamine infusion and was found to have no effect on gastric blood flow. This may be explained by the fact that gastric acid secretion was negligible under such experimental conditions. Secretion of hydrogen ions by the parietal cells is associated with movement of alkali into the interstitial fluid on an equimolar basis [I1 1. Kivilaakso et al [II] and Smith et al [22] have clearly shown that the actively secreting stomach is much more resistant to ulceration than the stomach that is inhibited by metiamide. This finding supports the concept that the alkaline tide is of importance in protecting the tissue against ulceration. Kivilaakso et al [II] reported that histamine stimulation caused a small, insignificant increase in the intramural pH of rabbit
27
Cheung and Sonnenschein
gastric mucosa. The inability to demonstrate a statistically significant increase in the intramural pH in rabbit gastric mucosa may be due to the fact that the mucosa cannot contain luminal hydrogen ions. As a result of hydrogen ions entering the tissue during histamine stimulation, the release of alkali into the interstitial fluid was acidified by the hydrogen ions entering the tissue and therefore, a significant increase in the intramural pH was not observed during histamine stimulation. In the present study we were able to demonstrate a significant increase in the intramural pH during histamine infusion in the dog stomach, which has a much greater ability than the rabbit stomach to contain acid within the lumen. We also found that cimetidine inhibition of acid secretion prevented such mucosal alkalinity, even in the presence of background histamine infusion. In recent controlled studies [6,7], it has been reported that cimetidine did not adequately protect seriously ill patients from acute upper gastrointestinal bleeding and that antacids were better for this purpose. The researchers suggested that the inefficacy of cimetidine in the prevention of acute stress ulcers may be due to several factors: It impairs the secretory state of the mucosa, decreases the buffering capacity of the mucosa, and inadequately reduces intraluminal acidity in some patients. The result of our study clearly supports the notion that cimetidine inhibition of acid secretion reduces the mucosal alkalinity and mucosal blood flow. Guth et al [23] demonstrated a strong correlation between the gastric acid secretory rate and mucosal blood flow as measured by carbon-14 aminopyrine clearance in healthy control subjects. If cimetidine totally inhibited the basal secretion (approximately 2 mEq/30 min), it would result in an approximately 30 percent reduction in mucosal blood flow. It is possible that the associated decrease in mucosal blood flow and mucosal alkalinity during cimetidine inhibition may contribute to its inefficacy in the prevention of severe erosion
in critically
ill patients.
Summary The effects of cimetidine inhibition of acid secretion on gastric mucosal blood flow and the intramural pH of gastric mucosa were assessed in a canine model ex vivo. Intravenous infusion of histamine at 1.0 pg/kg per minute resulted in brisk acid secretion, which was associated with an increase in total gastric and mucosal blood flow and the intramural pH of gastric mucosa. The increased blood flow and mucosal alkalinity may be compensatory mechanisms of the stomach to enhance its tolerance of luminal acid. Cimetidine inhibition of acid secretion was associated with a reduction in mucosal alkalinity and total gastric and mucosal blood flow. These adverse effects of cimetidine on blood flow and mucosal pH, two important factors in the stomach’s self-defense against
metidine 20
acid injury, may explain the inefficacy in the prevention of stress ulcers.
of ci-
References 1. lppoliti AF, Sturdevant RAL, lsenberg JI, et al. Cimetiiine versus intensive antacid therapy for duodenal ulcer. Gastroenterology 1978;74:393-5. 2. Dunn DH, Fischer RC, Silvis SE, Onstad GR, Howard RJ, Delaney JP. The treatment of hemorrhagic gastritis with cimetidine. Surg Gynecol Obstet 1978;147:737-9. 3. MacDonald AS, Steele BJ, Bottomley MG. Treatment of stress-induced upper gastrointestinal haemorrhage with metiamide. Lancet 1978; 1:88-70. 4. MacDougall BRD, Bailey RJ, Williams R. t-l2 receptor-antage nists and antacids in the prevention of acute gastrointestinal haemorrhage in fulminant hepatic failure: two controlled trials. Lancet 1977;1:617-9. 5. Jones RH, Rudge CJ, Bewick M, Parsons V, Weston MJ. Cimetidine: prophylaxis against upper gastrointestinal haemorrhage after renal transplantation. Br Med J 1978;l: 398-400. 6. Priebe HJ, Skillman JJ, Bushnell LS, Long PC, Silen W. Antacid versus cimetidine in preventing acute gastrointestinal bleeding. N Engl J Med 1980;302:426-30. 7. Zinner MI, Zuidema GD, Smith PL, Mignosa M. The prevention of upper gastrointestinal tract bleeding in patients in an intensive care unit. Surg Gynecol Obstet 1981;153:214-20. 8. Ritchie WP, Jr. Acute gastric mucosal damage induced by bile salts, acid and ischemia. Gastroenterology 1975;68:699707. 9. Moody FG, McGreevy J, Zalewsky C, Cheung LY, Simons M. The cytoprotective effect of mucosal blood flow in experimental erosive gastritis. Acta Physiol Stand (special supplement) 1978;35-43. 10. Kivilaakso E, Fromm D, Silen W. Relationship between ulceration and intramural pH of gastric mucosa during hemorrhagic shock. Surgery 1978;84:70-8. 11. Kivilaakso E, Fromm D, Silen W. Effect of the acid secretory state on intramural pH of rabbit gastric mucosa. Gastroenterology 1978;75:641-8. 12. Cheung LY. Effect of intraarterial infusion of bicarbonate on gastric intramural pH and ulceration. Surg Forum 1980; 31:117. 13. Cheung LY, Toenjes AA, Sonnenschein LA. Acidification of arterial blood enhances gastric mucosal injury induced by bile salts in dogs. Am J Surg 1982;143:74-9. 14. Konturek SJ, Tasler J, Obtulowicz W, Rehfeld JF. Effect of rnetiamide, a histamine Hfleceptor antagonsit, on mucosal blood flow and serum gastrin level. Gastroenterolmy 1974;66:982-6. 15. Delaney JP, Michel HM, Bond J. Cimetidine and gastric blood flow. Surgery 1978;84:190-3. 16. Moody FG, Durbin RP. Effects of glycine and other instillates on concentration of gastric acid. Am J Physiol 1965;209: 122-6. 17. Archibald LH. Moody FG, Simon MA. The measurement of gastric mucosal blood flow by radioactive microspheres. J Appl Physiol 1975;38:1051-6. 18. Tague LL, Jacobson ED. Evaluations of ‘C aminopyrine clearance for determination of gastric mucosal blood flow. Proc Sot Exp Biol Med 1976;151:707-10. 19. Jacobson ED, Linford RH, Grossman MI. Gastric secretion in relation to mucosal blood flow studied by a clearance tech nlque. J Clin Invest 1966;45:1:13. 20. Cheuncl LY, Moody FG, Larson K, Lowry SF. Oxygen consumbtion during-cimetidine and prostablandin E; inhibition of acid secretion. Am J Physiol 1978;234:445-50. 21. Cheung LY. Topical effects of 16, 16 dirnethyl prostaglandin EP on gastric blood flow in dogs. Am J Physiol 1980;238: G514-9. 22. Smith P, O’Brien P, Fromm 0, Silen W. Secretory state of
gastric mucosa and resistance to injury by exogenous acid. Am J Surg 1977;133:81. 23. Guth PH, Baumann H, Grossman MI. Aures D, Elashoff J. Measurement of gastric mucosal blood flow in man. Gas-
troenterology 1978;74:831-4. The American Journal of Surgery
Cimetidine and Gastric Blood Flow
Discussion Wallace Parks Ritchie (Charlottesville, VA): I enjoyed this paper very much. Drs. Cheung and Sonnenschein’s data provide a very clear demonstration, one of the first of which I am aware, that coincident with acid secretion, the alkaline tide can provide a buffer to the mucosa, and cimetidine vitiates this effect, although the pH achieved is never in the range associated wtih experimental ulcerogenesis. He suggested that this effect in combination with decreased blood flow may be responsible for the relative inefficacy of cimetidine as prophylaxis against stress ulceration. My own impression is the first factor that he mentioned; namely, cimetidine’s inability to maintain and achieve a high intraluminal pH is probably more important in that respect. I think the real implication of this study is the possibility that not only may cimetidine be inefficacious, but it may also be harmful in the setting in which a stress ulcer develops because of the two effects he points out. I think this is an important clinical concern because of the widespread use of both cimetidine and antacids in many intensive care units. I wonder if the authors will tell us whether they have any data to indicate that cimetidine has similar effects to those shown today in the setting which might simulate stress ulcer disease. David Fromm (Syracuse, NY): Drs. Cheung and Sonnenschein have confirmed observations made by several researchers. I would like to ask a few questions. First, did the arterial pH change at all during the study? Second, what is the explanation for the progressive increase in intramural pH during histamine stimulation? It took over an hour for the intramural pH to reach a plateau. Could this be due to venous congestion or edema that might occur as a result of using the plexiglass chamber? Finally, have the authors had the opportunity to confirm the observation of Kivalaakso et al, that the alkaline tide is indeed involved in the protection of the canine gastric mucosa against the harmful effects of bile salts? Edward T. Peter (Fresno, CA): Approximately 20 years ago we looked at the vascular effects of histamine on the stomach as well as many other agents, and generally found that all of those things that increased gastric secretion also increased mucosal blood flow. This was subsequently confirmed by Drs. Moody and Delaney. We also gave some diamine oxidase, which is histaminase that is only partially
Volume 145, January 1993
purified, but we negated the vascular effects as well as the secretory effects of the histamine. This was subsequently used by my colleagues in physiology to study pulmonary circulation, and the same thing occurred. We postulated that there were some receptors within the vascular circuit that responded to histamine which could not be blocked by conventional antihistamines but could be blocked by diamine oxidase or enzymatically by inactivating the histamine. It is very interesting that cimetidine has the effect of blocking this, and we now see that there are Hz receptors in the arterioles most likely. I wonder if you have studied other circuits such as the pulmonary circuit and the reaction to cimetidine. Frank G. Moody (Salt Lake City, UT): The authors might inform us as to whether they have used other inhibitors, or what happens when you discontinue the histamine in terms of the intramural pH. Laurence Y. Cheung (closing): To answer Dr. Ritchie’s question, I really have not carried out any study of the effect of cimetidine on the severity of ulcer formation, and whether it aggravates the ulcer or protects it. On the other hand, Dr. Silen’s laboratory has performed several studies that have indicated that metiamide in those experimental conditions makes the ulceration worse and that histamine stimulation protects it. Dr. Fromm, we did measure arterial pH at least during the 2 hour study, and we did not find any changes in the arterial pH. We did find that the gastric venous pH increased with histamine stimulation, perhaps because of the bicarbonate release. It is not significant enough, at least during the short period of the experiment, to raise systemic arterial pH. I did not do the experiment, as Kivilaakso did, to measure the effect of cimetidine on bile injury to the stomach. We did not measure any blood flow other than gastric blood flow. When we stopped the histamine infusion the intramural pH returned to 7.4. When we used prostaglandin we found that prostaglandin inhibition of acid secretion reduced the intramural pH to a value even lower than that achieved with cimetidine. We do not know why prostaglandin lowers the mucosal pH even more than cimetidine, to about 7.25 or 7.3. It might be related to the fact that prostaglandin not only inhibits acid secretion but also stimulates alkaline secretion. When there is alkaline secretion maybe there is acid released into the mucosa.
29
Laurence Y. Cheung, MD, St. Louis, Missouri Leonard A. Sonnenschein, BS, St. Louis, Missouri
Cimetidine, an Hz-receptor antagonist, is widely used to inhibit acid secretion in patients with chronic duodenal or gastric ulcers [I]. However, its efficacy in the prevention and treatment of acute upper gastrointestinal bleeding in severely ill patients is uncertain [2-71. Since gastric blood flow [8,9] and the intramural pH of the gastric mucosa [IO--131 play an essential role in mucosal tolerance to acute ulcerogenesis, it is desirable to know the effects of cimetidine on the gastric microcirculation and intramural PH. Konturek et al [14] demonstrated in dogs that metiamide inhibition of acid secretion stimulated by several secretogogues was associated with a significant reduction in mucosal blood flow as measured by aminopyrine clearance. Delaney et al [15] reported that cimetidine caused no change in total gastric and mucosal blood flow as determined by radioactive microspheres to any portion of the stomach in unstimulated dogs. The discrepancy between these two reports has been attributed to variables in the methods of measuring mucosal blood flow and to the different secretory states of the canine stomach when H2-receptor antagonists were administered [15]. Therefore, this study was designed to compare the effect of cimetidine on mucosal blood flow as determined by microspheres with that measured by carbon-14 aminopyrine clearance. In addition, the effect of secretory inhibition by cimetidine on the intramural pH of the gastric mucosa was also evaluated. From the Surgical Service of the Veterans Administration Hospital, and the Department of Surgery, Washington University School of Medicine, St. Louis, Missouri. This investigation was supported by the Medical Research Service of the Veterans Administration and by grant AM25998-03 from the National institute of Health, Bethesda, Maryland. Requests for reprints should be addressed to Laurence Y. Cheung, MO, Department of Surgery, Washington University School of Medicine, 4960 Audubon Avenue, St. Louis, Missouri 63110. Presented at the 23rd Annual Meeting of the Society for Surgery of the Alimentary Tract, Chicago, Illinois, May 18-19, 1982.
24
Material
and Methods
Mongrel dogs that weighed from 20 to 25 kg were anesthetized with sodium pentobarbitol (25 mg/kg) and maintained on a Harvard respirator throughout each experiment. Polyvinyl catheters (inside diameter (ID) 0.058 inch) were placed in both femoral veins for infusion of fluid, blood, and drugs. Arterial pressure was monitored through a catheter inserted into the left femoral artery (Statham transducer P23AA; Hewlett Packard direct writing recorder). A catheter (ID 0.067 inch) was placed in the left ventricle through the right carotid artery. A chambered segment of gastric corpus with an isolated vascular pedicle was prepared as described by Moody and Durbin [16]. This chamber provided a mucosal surface of approximately 36 cm2. In all experiments, isotonic hydrogen chloride solution (0.15N) was placed into and recovered from the chamber at 15 minute intervals. The secretory volume was calculated as the change in weight between the instilled and recovered solutions. Hydrogen ion flux was determined by potentiometric titration of instillate and recovered samples to a pH of 6.5 (Radiometer, Copenhagen) with O.lN sodium hydroxide. Venous flow through the gastric segment was measured by occluding the gastrosplenic vein proximal to a Silastic@ catheter (ID 0.56 inch) placed in the superior splenic vein. Venous pressure was continuously monitored by a polyvinyl catheter (ID 0.058 inch) placed in a terminal splenic vein (Statham transducer P23BB). For each 15 minute period, timed venous samples were collected at baseline pressure in preweighed tubes containing EDTA. Volume was calculated on the basis of 1 g/ml. Details of the technique of measuring total gastric and mucosal blood flow with y-labeled microspheres have been reported previously by Archibald et al [17]. In brief, three measurements were made in each dog by injecting microspheres labeled with strontium-85, cerium-141, or chromium-51. The first injection was given during a rest period. The second and third injections were given during the steady state period of histamine-stimulated acid secretion and cimetidine inhibition. For each injection, approximately 100,000 to 500,000 spheres of 15 f 5 ~1in diameter were injected from a mixing chamber into the left ventricle.
The American
Journal of Surgery
Cimetidine and Gastric Blood Flow
A reference sample was drawn at 10 ml/min from the distal aorta for 80 seconds, beginning 10 seconds before injection. The amount of blood loss from arterial and venous sampling was replaced by transfusion. After the completion of each experiment, the gastric mucosa, submucosa, and muscularis were bluntly separated and dried. The tissue specimens were dissolved in 15 percent ethanolic potassium hydroxide solution, centrifuged, and then counted in a gamma counter. Flow was calculated by comparing the activity of each isotope in the tissue with that in the reference sample. Carbon-14 aminopyrine clearance was measured as described by Tague and Jacobson [18]. Carbon-14 aminopyrine (Amersham-Searle, 52.8 @Zi/mg) in a loading dose of 0.15 &i/kg was injected intravenously over 2 minutes. This was followed by continuous intravenous infusion of carbon-14 aminopyrine (0.08 &i/kg per hour). Each animal was equilibrated with aminopyrine for 1 hour before starting the study. At hourly intervals, 10 ml blood samples were drawn into heparinized tubes from the femoral arterial catheter and centrifuged at 4’C to obtain plasma. Mucosal bathing solution was instilled and recovered at 15 minute intervals. One ml of gastric bathing solution was pipetted directly into a scintillation vial and mixed with 10 ml of phase combining system (Amersham-Searle). Plasma samples were extracted as described by Tague and Jacobson [18] and mixed with 15 ml of phase combining system for cell counting. Appropriate blanks were also counted. Gastric and plasma samples were counted for 10 minutes. After subtraction of blanks, cpm/ml was determined and the following equation used to calculate clearance: clearance ml/min = cpm/ml gastric juice/cpm/ml plasma X ml/min gastric juice secreted plus bathing fluid. The intramural pH was measured using a miniature pH electrode (Microelectrode Inc., Londonderry, NH) as decribed by Kivilaakso et al [10,11]. An area of 0.5 to 1.0 cm2 of the gastric wall was denuded of its seromuscular coat. The electrode was directly advanced from the serosal surface through the denuded area into the underlying mucosa at a micrometer depth of 500 p using a micromanipulator (Narishige, Tokyo). The denuded area was
coated with a drop of sesame oil to prevent excessive evaporation and carbon dioxide loss. A calomel reference electrode was connected to the serosal surface of the chamberthrough a saline-agarbridge.The millivoltagewas recorded by a high input impedance pH electrometer (Radiometer).Measurementsof intramuralpH were made continuously throughout each experiment. The average millivoltage was converted to pH units using a calibration curve. The microelectrodes were calibrated in a series of nonphosphate buffers at 37V. The pH response curve was strictly linear, at least over a pH range of 5 to 9. The approximate location of the microelectrode in the gastric wall during the pH measurements was studied at the end of experiments in several dogs by generating a lesion around the tip of the electrode by electrocoagulation. The tissue damage was identified by a pathologist with light microscopy. All of the identified lesions were situated in the deep third layer of the mucosa near the muscularis mucosa. After three resting periods, histamine was infused intravenously at 1.0 pg/kg per minute for the remaining 14 periods. The acid secretion usually reached a plateau 4 periods after the beginning of histamine infusion (period 7) and remained at a steady state throughout the experi-
Volume 145,
January 1983
IV Ctmetldme IV Hlstomtne
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Figure 1. Sequenttal changes in secretory votims and total ackt output In six expertlments, expressed as ths maan f standard enorofths-HkstanVne wask&ssdtntravenousQat ipg/kg per minute. Cknatkikts was given tntravenousty (50 pglkg per mbnde) In addttb to the background htstamhm.
ment. Cimetidine (50 pg/kg per minute) was added intravenously to the background histamine infusion from periods 10 through 13. The dose chosen for cimetidine was the minimum that would consistently give nearly 100 percent inhibition of histamine-stimulated acid secretion. The results from each period were pooled and represented by mean plus or minus the standard error of the mean. The data was analyzed with the paired Student’s t test.
Results The sequential changes in the acid secretion and secretory volume are shown in Figure 1. Before histamine infusion, the secretory volume was negligible. In fact, there was a small net hydrogen ion loss from the lumen as calculated by the change in the amount of hydrogen ion between the instilled and recovered solutions. Intravenous infusion of histamine stimulated the total acid secretion as well as the volume (p
Cheung and Sonnenschein
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PERIODS (I5 MINI F&we 3. Seqnwtlal changes h cahm-14 amhqyrbe chwrame and mucosal blood flow (n = 8) as detemdnedby mkroepheres
PERIODS (I5 MINI [email protected]~hl-nowandtotalgastrkbhmd flowmeasomdbymkmqhms &lfngpl9rws3,9,aml13hflw experhents, expressed as the mean f standard error of the
mean. Htstamh waskhsedhtrawmouslyat 1pgikgwmhte. Clmetidhe was g&n intravenous/y(SO pg/kg per mhte) addMon to backgiwnd hlatamkte.
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trated. Intravenous infusion of histamine resulted in a significant increase in total venous flow (p <0.05) and total gastric blood flow as determined by microspheres (p <0.02). Cimetidine inhibition of acid secretion was accompanied by a reduction in total gastric blood flow as measured by venous flow (p <0.02) and by microspheres (p <0.05). Paired t test analysis showed no significant difference between the total gastric blood flow of cimetidine-inhibited periods and that of the prestimulated periods. Total gastric blood flow measured simultaneously by venous outflow correlated well with microsphere-determined blood flow. The linear regression revealed a highly significant correlation (r = 0.91, slope = 0.97, n = 15, p
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duringperiods 3, g, and 12, expressedas the mean f standard emwofthemean. Hlstamhe washhsedhtraveaus&at 1Wkg per minute. Cknettdhe was given Intravenously( 50 pgf kg per m/note) In addtth to backgrwmd hlstamlne.
(p <0.02). Despite the background histamine infusion, cimetidine inhibition of acid secretion was accompanied by a significant reduction in carbon-14 aminopyrine clearance and microsphere-determined mucosal blood flow. Mucosal blood flow during the cimetidine inhibition period (period 13) was not significantly different from that in the prestinudated period (period 3). In Figure 4 the percent increase in blood flow during histamine infusion and the percent reduction in blood flow after cimetidine inhibition of acid secretion are shown. Histamine infusion resulted in a significant increase in total blood flow as measured by venous flow and microspheres and mucosal blood flow as indicated by microspheres and carbon-14 aminopyrine clearance (p cO.05). The degree of increase in mucosal blood flow was slightly, but not significantly, higher than total gastric blood flow. Cimetidine inhibition resulted in a significant reduction (p <0.05) in total gastric blood flow and mucosal blood flow as measured by all these methods. There was no significant difference between cimetidine-inhibited periods and prestimulated periods in any measurement, which indicated that total and
The American Journal of Surgery
Cimetidine and Gastric Blood Flow
I.V. Cimetidlne
I.\/: Histamine
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0 IA 0 Flgore 4. Percent changesIn blood flow duringhktamlne sflmuIatkn and clmetldlne Inhlbltlonof add secretton.All data were expressed as the mean l standard error of the mean of five experlments except canbon-14 amlnopyrlneclearance ( n = 8).
mucosal blood flow returned to the preinfusion level when secretion was inhibited by cimetidine. In Figure 5 the sequential changes of the intramural pH of gastric mucosa during histamine stimulation and cimetidine inhibition are shown. In control dogs, the intramural pH of the gastric mucosa increased significantly (p
Valume 145, January 1333
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PERIODS (I5 MINI Figure 5. Effects of intravenoushfstamfneand clmetldlne on the lntramwralpH of the gastrk nucosa. ln conttvl &gs, intravenous hlstamlne slgnlfkantly increased the intramuralpH ( p < 0.00 1) when compared with psrkds before hlstamlneInfusion.llm addMonof clmsthilneto backgmundh&am&m sWfkant& reduced the Intramural pH (p <0.007) when compared wlth hlstamlne lnfuslonakne.
drochloric acid requires the utilization of energy and oxygen. Since such a metabolic demand is no longer present during cimetidine inhibition [20], one would expect that gastric blood flow would return to the resting level. The effect of a secretory inhibitor on gastric microcirculation may vary depending on whether it is administered during resting or during stimulated conditions [21]. In the study reported by Delaney et al [15] cimetidine was administered in anesthetized dogs without the background histamine infusion and was found to have no effect on gastric blood flow. This may be explained by the fact that gastric acid secretion was negligible under such experimental conditions. Secretion of hydrogen ions by the parietal cells is associated with movement of alkali into the interstitial fluid on an equimolar basis [I1 1. Kivilaakso et al [II] and Smith et al [22] have clearly shown that the actively secreting stomach is much more resistant to ulceration than the stomach that is inhibited by metiamide. This finding supports the concept that the alkaline tide is of importance in protecting the tissue against ulceration. Kivilaakso et al [II] reported that histamine stimulation caused a small, insignificant increase in the intramural pH of rabbit
27
Cheung and Sonnenschein
gastric mucosa. The inability to demonstrate a statistically significant increase in the intramural pH in rabbit gastric mucosa may be due to the fact that the mucosa cannot contain luminal hydrogen ions. As a result of hydrogen ions entering the tissue during histamine stimulation, the release of alkali into the interstitial fluid was acidified by the hydrogen ions entering the tissue and therefore, a significant increase in the intramural pH was not observed during histamine stimulation. In the present study we were able to demonstrate a significant increase in the intramural pH during histamine infusion in the dog stomach, which has a much greater ability than the rabbit stomach to contain acid within the lumen. We also found that cimetidine inhibition of acid secretion prevented such mucosal alkalinity, even in the presence of background histamine infusion. In recent controlled studies [6,7], it has been reported that cimetidine did not adequately protect seriously ill patients from acute upper gastrointestinal bleeding and that antacids were better for this purpose. The researchers suggested that the inefficacy of cimetidine in the prevention of acute stress ulcers may be due to several factors: It impairs the secretory state of the mucosa, decreases the buffering capacity of the mucosa, and inadequately reduces intraluminal acidity in some patients. The result of our study clearly supports the notion that cimetidine inhibition of acid secretion reduces the mucosal alkalinity and mucosal blood flow. Guth et al [23] demonstrated a strong correlation between the gastric acid secretory rate and mucosal blood flow as measured by carbon-14 aminopyrine clearance in healthy control subjects. If cimetidine totally inhibited the basal secretion (approximately 2 mEq/30 min), it would result in an approximately 30 percent reduction in mucosal blood flow. It is possible that the associated decrease in mucosal blood flow and mucosal alkalinity during cimetidine inhibition may contribute to its inefficacy in the prevention of severe erosion
in critically
ill patients.
Summary The effects of cimetidine inhibition of acid secretion on gastric mucosal blood flow and the intramural pH of gastric mucosa were assessed in a canine model ex vivo. Intravenous infusion of histamine at 1.0 pg/kg per minute resulted in brisk acid secretion, which was associated with an increase in total gastric and mucosal blood flow and the intramural pH of gastric mucosa. The increased blood flow and mucosal alkalinity may be compensatory mechanisms of the stomach to enhance its tolerance of luminal acid. Cimetidine inhibition of acid secretion was associated with a reduction in mucosal alkalinity and total gastric and mucosal blood flow. These adverse effects of cimetidine on blood flow and mucosal pH, two important factors in the stomach’s self-defense against
metidine 20
acid injury, may explain the inefficacy in the prevention of stress ulcers.
of ci-
References 1. lppoliti AF, Sturdevant RAL, lsenberg JI, et al. Cimetiiine versus intensive antacid therapy for duodenal ulcer. Gastroenterology 1978;74:393-5. 2. Dunn DH, Fischer RC, Silvis SE, Onstad GR, Howard RJ, Delaney JP. The treatment of hemorrhagic gastritis with cimetidine. Surg Gynecol Obstet 1978;147:737-9. 3. MacDonald AS, Steele BJ, Bottomley MG. Treatment of stress-induced upper gastrointestinal haemorrhage with metiamide. Lancet 1978; 1:88-70. 4. MacDougall BRD, Bailey RJ, Williams R. t-l2 receptor-antage nists and antacids in the prevention of acute gastrointestinal haemorrhage in fulminant hepatic failure: two controlled trials. Lancet 1977;1:617-9. 5. Jones RH, Rudge CJ, Bewick M, Parsons V, Weston MJ. Cimetidine: prophylaxis against upper gastrointestinal haemorrhage after renal transplantation. Br Med J 1978;l: 398-400. 6. Priebe HJ, Skillman JJ, Bushnell LS, Long PC, Silen W. Antacid versus cimetidine in preventing acute gastrointestinal bleeding. N Engl J Med 1980;302:426-30. 7. Zinner MI, Zuidema GD, Smith PL, Mignosa M. The prevention of upper gastrointestinal tract bleeding in patients in an intensive care unit. Surg Gynecol Obstet 1981;153:214-20. 8. Ritchie WP, Jr. Acute gastric mucosal damage induced by bile salts, acid and ischemia. Gastroenterology 1975;68:699707. 9. Moody FG, McGreevy J, Zalewsky C, Cheung LY, Simons M. The cytoprotective effect of mucosal blood flow in experimental erosive gastritis. Acta Physiol Stand (special supplement) 1978;35-43. 10. Kivilaakso E, Fromm D, Silen W. Relationship between ulceration and intramural pH of gastric mucosa during hemorrhagic shock. Surgery 1978;84:70-8. 11. Kivilaakso E, Fromm D, Silen W. Effect of the acid secretory state on intramural pH of rabbit gastric mucosa. Gastroenterology 1978;75:641-8. 12. Cheung LY. Effect of intraarterial infusion of bicarbonate on gastric intramural pH and ulceration. Surg Forum 1980; 31:117. 13. Cheung LY, Toenjes AA, Sonnenschein LA. Acidification of arterial blood enhances gastric mucosal injury induced by bile salts in dogs. Am J Surg 1982;143:74-9. 14. Konturek SJ, Tasler J, Obtulowicz W, Rehfeld JF. Effect of rnetiamide, a histamine Hfleceptor antagonsit, on mucosal blood flow and serum gastrin level. Gastroenterolmy 1974;66:982-6. 15. Delaney JP, Michel HM, Bond J. Cimetidine and gastric blood flow. Surgery 1978;84:190-3. 16. Moody FG, Durbin RP. Effects of glycine and other instillates on concentration of gastric acid. Am J Physiol 1965;209: 122-6. 17. Archibald LH. Moody FG, Simon MA. The measurement of gastric mucosal blood flow by radioactive microspheres. J Appl Physiol 1975;38:1051-6. 18. Tague LL, Jacobson ED. Evaluations of ‘C aminopyrine clearance for determination of gastric mucosal blood flow. Proc Sot Exp Biol Med 1976;151:707-10. 19. Jacobson ED, Linford RH, Grossman MI. Gastric secretion in relation to mucosal blood flow studied by a clearance tech nlque. J Clin Invest 1966;45:1:13. 20. Cheuncl LY, Moody FG, Larson K, Lowry SF. Oxygen consumbtion during-cimetidine and prostablandin E; inhibition of acid secretion. Am J Physiol 1978;234:445-50. 21. Cheung LY. Topical effects of 16, 16 dirnethyl prostaglandin EP on gastric blood flow in dogs. Am J Physiol 1980;238: G514-9. 22. Smith P, O’Brien P, Fromm 0, Silen W. Secretory state of
gastric mucosa and resistance to injury by exogenous acid. Am J Surg 1977;133:81. 23. Guth PH, Baumann H, Grossman MI. Aures D, Elashoff J. Measurement of gastric mucosal blood flow in man. Gas-
troenterology 1978;74:831-4. The American Journal of Surgery
Cimetidine and Gastric Blood Flow
Discussion Wallace Parks Ritchie (Charlottesville, VA): I enjoyed this paper very much. Drs. Cheung and Sonnenschein’s data provide a very clear demonstration, one of the first of which I am aware, that coincident with acid secretion, the alkaline tide can provide a buffer to the mucosa, and cimetidine vitiates this effect, although the pH achieved is never in the range associated wtih experimental ulcerogenesis. He suggested that this effect in combination with decreased blood flow may be responsible for the relative inefficacy of cimetidine as prophylaxis against stress ulceration. My own impression is the first factor that he mentioned; namely, cimetidine’s inability to maintain and achieve a high intraluminal pH is probably more important in that respect. I think the real implication of this study is the possibility that not only may cimetidine be inefficacious, but it may also be harmful in the setting in which a stress ulcer develops because of the two effects he points out. I think this is an important clinical concern because of the widespread use of both cimetidine and antacids in many intensive care units. I wonder if the authors will tell us whether they have any data to indicate that cimetidine has similar effects to those shown today in the setting which might simulate stress ulcer disease. David Fromm (Syracuse, NY): Drs. Cheung and Sonnenschein have confirmed observations made by several researchers. I would like to ask a few questions. First, did the arterial pH change at all during the study? Second, what is the explanation for the progressive increase in intramural pH during histamine stimulation? It took over an hour for the intramural pH to reach a plateau. Could this be due to venous congestion or edema that might occur as a result of using the plexiglass chamber? Finally, have the authors had the opportunity to confirm the observation of Kivalaakso et al, that the alkaline tide is indeed involved in the protection of the canine gastric mucosa against the harmful effects of bile salts? Edward T. Peter (Fresno, CA): Approximately 20 years ago we looked at the vascular effects of histamine on the stomach as well as many other agents, and generally found that all of those things that increased gastric secretion also increased mucosal blood flow. This was subsequently confirmed by Drs. Moody and Delaney. We also gave some diamine oxidase, which is histaminase that is only partially
Volume 145, January 1993
purified, but we negated the vascular effects as well as the secretory effects of the histamine. This was subsequently used by my colleagues in physiology to study pulmonary circulation, and the same thing occurred. We postulated that there were some receptors within the vascular circuit that responded to histamine which could not be blocked by conventional antihistamines but could be blocked by diamine oxidase or enzymatically by inactivating the histamine. It is very interesting that cimetidine has the effect of blocking this, and we now see that there are Hz receptors in the arterioles most likely. I wonder if you have studied other circuits such as the pulmonary circuit and the reaction to cimetidine. Frank G. Moody (Salt Lake City, UT): The authors might inform us as to whether they have used other inhibitors, or what happens when you discontinue the histamine in terms of the intramural pH. Laurence Y. Cheung (closing): To answer Dr. Ritchie’s question, I really have not carried out any study of the effect of cimetidine on the severity of ulcer formation, and whether it aggravates the ulcer or protects it. On the other hand, Dr. Silen’s laboratory has performed several studies that have indicated that metiamide in those experimental conditions makes the ulceration worse and that histamine stimulation protects it. Dr. Fromm, we did measure arterial pH at least during the 2 hour study, and we did not find any changes in the arterial pH. We did find that the gastric venous pH increased with histamine stimulation, perhaps because of the bicarbonate release. It is not significant enough, at least during the short period of the experiment, to raise systemic arterial pH. I did not do the experiment, as Kivilaakso did, to measure the effect of cimetidine on bile injury to the stomach. We did not measure any blood flow other than gastric blood flow. When we stopped the histamine infusion the intramural pH returned to 7.4. When we used prostaglandin we found that prostaglandin inhibition of acid secretion reduced the intramural pH to a value even lower than that achieved with cimetidine. We do not know why prostaglandin lowers the mucosal pH even more than cimetidine, to about 7.25 or 7.3. It might be related to the fact that prostaglandin not only inhibits acid secretion but also stimulates alkaline secretion. When there is alkaline secretion maybe there is acid released into the mucosa.
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