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Mucosal Blood Flow in Canine Antral and Fundic Pouches
Vol. 60, No.2 Printed in U. S. A.
GASTROENTEROLOGY
Copyright
©
1971 by The Williams & Wilkins Co.
MUCOSAL BLOOD FLOW IN CANINE ANTRAL AND FUNDIC POUCHES JACK RUDICK, M.D., J. LAWRENCE WERTHER, M.D., MARK L. CHAPMAN, M.D., DAVID A. DREILING, M.D., AND HENRY D. JANOWITZ, M.D.
Department of Surgery and the Division of Gastroenterology. The Mount Sinai School of Medicine of the City University of New York, New York, New York
Aminopyrine clearance was used to compare mucosal blood flow in the resting and stimulated fundus and antrum. The intraluminal instillation of 0.16 N Hel provided a pH gradient allowing aminopyrine to move into the lumen. Resting mucosal perfusion was 0.64 ± 0.23 (so) ml per min per g of mucosa for the fundus and 0.34 ± 0.10 ml per min per g of wet mucosa for the antrum. Extrapolated mucosal blood flow of the resting fundus by plotting aminopyrine clearance against acid output during secretory stimulation correlated well with mucosal blood flow during acid instillations. Graded doses of histamine by intravenous infusion stimulated mucosal blood flow in the fundus parallel to acid secretion; a maximal dose (2 Ilg of base per kg per min) increased mucosal perfusion of the fundus 4-fold and doubled antral mucosal perfusion. Isoproterenol (1 Ilg per kg per min intravenously) increased mucosal blood flow to the resting fundus by 100% and to the antrum by 110%. Vasopressin (4 mU per kg per min intravenously) decreased resting fundic mucosal blood flow by 75% and antral blood flow by 80%. The present study lends further support to the validity of aminopyrine clearance as a measure of mucosal blood flow. It is further shown that: (1) aminopyrine clearance in the fundus and antrum is not dependent on the secretion of hydrogen ions and can be measured by using exogenous acid; (2) no redistribution of mucosal blood flow from antrum to corpus occurs with histamine stimulation; (3) primary alterations in mucosal blood flow can be induced in both pouches by vasoactive drugs without affecting acid secretion. Recent innovations in methodology 2-10 have revived interest in the measurement of gastric blood flow. Most studies, however, have concentrated either on the Received July 1, 1970. Accepted September 8, 1970. This work was presented in part to the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, April 18, 1969 and an abstract has appeared. 1 Address requests for reprints to: Dr. Jack Rudick, Department of Surgery, The Mount Sinai Hospital, 3 East l00th Street, New York, New York 10029. This work was supported by United States Public Health Service Grant AM-11580 from the National Institutes of Arthritis and Metabolic Diseases. Dr. Rudick is a recipient of Career Scientist Award 1-541 from the New York Health Research Council.
whole stomach or on the oxyntic gland area. Within certain limitations, the evidence points to a general relationship between gastric blood flow and acid secretion. Aminopyrine clearance from the gastric mucosa has been confirmed as a reliable method for the measurement of mucosal blood flow. 6 - 1o This method is dependent upon a pH gradient for aminopyrine to move into gastric juice. Since a pH in the gastric lumen less than the pK of aminopyrine (pK 5) is required, little information exists on mucosal blood flow in the antrum (a non-acid secreting portion of the stomach) or on the unstimulated corpus. In this study we have utilized exogenous 263
RUDICK ET AL.
264
acid to provide a pH gradient between plasma and gastric lumen. Aminopyrine clearance has thus been measured from the antrum and compared to the fundus. We have also attempted to determine whether alterations in blood flow are associated with fluxes of ions across the mucosa.
Methods Eight dogs of both sexes ranging in weight from 18 to 28 kg were used . Four dogs were prepared with vagally denervated antral and vagally denervated fundic pouches, 2 dogs with antral pouches only, and 2 dogs with fundic pouches alone. Pure antral pouches were obtained by delineating the antrum-corpus junction colorimetrically with 1 % toluidine blue intravenously.ll A 2-cm rim of tissue at the intermediate zone between antrum and corpus was excised to ensure a pure antral pouch preparation . This was subsequently confirmed by (a) histological examination of the intermediate zone and (b) monitoring antral pH during the intravenous administration of histamine (2 ILg of base per kg per min). Each pouch was equipped with a nylon-coated stainless steel gastric cannula which was exteriorized in the midline to assist complete emptying of the pouch when the animals stood upright during the test. The animals remained in good health and maintained stable weights during the period of study. At least 4 weeks elapsed before experiments were started. Experiments were conducted after the dogs had been deprived of food but not water of 18 hr prior to each observation. Each dog was studied no more than once a week. The animals were placed in a modified Pavlov frame and 0.9% saline was infused intravenously at a constant rate (0.6 ml per min). A loading dose of aminopyrine (20 mg per kg) was injected over 10 min, and was followed by a maintenance dose of 5 mg per kg hr throughout each experiment. The intralumenal instillation of 0.16 N HCI provided a pH gradient allowing aminopyrine to migrate into the lumen of each pouch. The volumes of acid solutions instilled were determined individually for each pouch. One end of a polyethelene tube was attached to an adaptor screwed on tightly into the gastric cannula and the other end was attached to a three-way stopcock. Antral pouches were filled to a hydrostatic pressure of 15 cm, and fundic pouches to a hydrostatic pressure of 6 cm. Capacities of the antral pouches ranged from 18 to 35 ml and the fundic pouches from
Vol . 60, No . 2
60 to 100 ml. During determinations of the pouch volumes and in subsequent experiments it was demonstrated that no leak was present. At the beginning of each experiment, each pouch was washed out with 0.16 N HCI warmed to 37 C, and the pouch was drained as completely as possible. The predetermined volume of acid with a nonabsorbable marker (4 mg per 100 ml of phenol red) was then introduced into the pouch and, after mixing, a 3-ml aliquot was removed to determine the residual volume. The acid solution was allowed to remain in the pouch for 15 min and then emptied by gravity drainage and gentle massage over the pouch area; a fresh solution was then introduced. Residual volumes were determined in the manner previously described. 12 Blood samples were obtained from a peripheral leg vein every 30 min. The volume of fluid recovered from each pouch was determined to the nearest 0.1 ml. Specimens were analyzed individually for volume, acidity, sodium, potassium, chloride, phenol red, osmolality, and aminopyrine concentration. Acidity was measured by titration to end point pH 7.0 with a pH-stat titrator (Radiometer TIT1c). Sodium, potassium, and chloride were determined with a Technicon AutoAnalyzer and osmolality with an advanced osmometer. Phenol red determinations were performed according to the method of Hunt. IS Aminopyrine clearance was determined by the method of Shore et al. 1. as adapted by Jacobson et al. 6 to measure mucosal plasma flow . Pouch contents and plasma were analyzed individually for aminopyrine concentration by the method of Brodie and Axelrod. 1 ' Mucosal blood flow in both pouches was measured: (a) in the resting state; (b) in response to graded doses of histamine, 0.25 to 2.0 ILg of base per kg per min; (c) in response to isoproterenol (Isuprel, Winthrop-Stearns, Inc.), 1 ILg per kg per min, and to Vasopressin (Parke, Davis and Co.), 4 m U per kg per min. In order to allow comparison with other studies (utilizing a variety of techniques), all of the pouches were excised after termination of the studies. The pouches were excised with the animals under general anesthesia and the gross fat was trimmed off. The mucosa was stripped from the underlying submucosa, gently blotted free of gross moisture, and weighed while wet. Mucosa of antrum was 33.0 ± 2.5% (so) of the pouch weight, while the mucosa of the fundus was 47.8 ± 4.2 % of the pouch weight. The weight of the mucosa of the pouches did not correlate with body weight because the size of the pouch was dependent not only on the size
of the dog (and stomach) but also on technical considerations, so that a larger animal may have had a smaller pouch. The results are therefore expressed as a function of mucosal weight rather than weight of the animal. Plasma clearance of aminopyrine was derived from the formula: GV c=---P X t X w
where C is plasma aminopyrine clearance in milliliters per min per g of wet mucosa; G is concentration of aminopyrine in gastric contents; V is volume of gastric contents at the end of each instillation period; P is plasma aminopyrine concentration; t is time in minutes; and w is weight of wet mucosa. Secretion of acid in the histamine experiments was expressed as microequivalents per g of wet mucosa per min. The surface area of the mucosa was measured by planimetry, enabling ionic movements to be determined per unit area (square centimeters). During the intravenous administration of aminopyrine in 2 of the dogs with double pouches, either 0.16 N HCI or isotonic mannitol (5.82 per 100 ml at pH 6.8 was instilled into each pouch; recovery of aminopyrine was compared when the pouches were acidified or neutral. In separate experiments in these 2 animals, 50 mg of aminopyrine dissolved in 0.16 N HCI were introduced into each pouch and the aminopyrine recovered in the pouch contents was determined at the end of 15 min. During the latter experiments, no aminopyrine was administered intravenously.
Results Recovery of aminopyrine. Recovery of aminopyrine from acidified antral and fun1. Aminopyrine recovery from antral and fundic pouches when acid (0 .16 N HCl) or neutral (mannitol) solutions were instilled into each pouch"
TABLE
Aminopyrine recovered Solution instilled
n'
I
~--------.----------
Antrum
Fundus
mg/I5 min
0.16N HCL ... Mannitol ...
265
MUCOSAL BLOOD FLOW
February 1971
35 1.12 ± 0.28·13.12 ± 0.52 33 0.19 ± 0.0510.52 ± 0.18
• Results are given as mean ± b n, no. of observations.
SD.
TABLE
2. Plasma aminopyrine clearance in resting
antral and fundic pouches' Dog
Antrum
Fundus
mllmin/g wet mucosa
1 2 3 4 5 6 7 8
Mean
0.22 0.46 0 . 21 0 . 39 0 . 43 0 . 31
± ± ± ± ± ±
0.08 0 .06 0.07 0.13 0.10 0.03
0.34 ± 0.10
• Results are given as mean ±
0.84 0 . 66 0.48 0.83 0 .50 0.58
± ± ± ± ± ±
0.17 0.21 0 .09 0 . 15 0 .08 0.16
0.64 ± 0.23
SD.
dic pouches in 2 dogs is seen in table 1. When isotonic mannitol at pH 6.8 instead of acid was instilled it resulted in a much reduced recovery of aminopyrine from each pouch, despite the maintenance of a constant plasma aminopyrine level. When 50 mg of aminopyrine dissolved in 0.16 N Hel were introduced into each pouch in the absence of circulating aminopyrine, recovery was 96.7 ± 3.6% (SE) for antral pouches and 97.8 ± 2.9% for fundic pouches. No significant absorption of aminopyrine was detected from the lumen of acidified pouches. In contrast, aminopyrine dissolved in mannitol at pH 6.8 almost completely disappeared from each pouch in 5 to 10 min. Resting mucosal blood flow in unstimulated antral and fundic pouches. Mean perfusion was 0.34 ± 0.10 (mean ± so) ml per min per g of wet mucosa for the antrum and 0.64 ± 0.23 ml per min per g of wet mucosa for the corpus. Individual values of each dog are seen in table 2. These figures are derived from resting values when aminopyrine clearance had stabilized. The perfusion ratio of antrum to corpus is thus 0.53: 1. It was found that it usually took about 45 to 60 min for the concentration of aminopyrine to reach a plateau in the pouch contents. Mean results from six experiments in 3 dogs are illustrated in figure 1. Antral aminopyrine clearance of 0.32 ± 0.06 ml per min per g of wet mucosa was 22% lower than a simi-
266
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lar instillation period after a plateau had been reached. Similarly, corpus aminopyrine clearance of 0.58 ± 0.08 ml per min per g of wet mucosa was 27% less than when perfusion had stabilized. To test further the validity of the clearance method, the length of instillation periods was varied. Once a plateau had been reached there was no difference in the perfusion per unit time when the instillation periods were varied from 15 to 30 or 60 min. It is also evident from figure 1 that there was no decline in aminopyrine clear-
ance with the passage of tIme, provided that plasma aminopyrine concentration remained stable. This is of interest since, with the passage of time there was frequently a greater loss of hydrogen ions from the antral pouches than at the beginning of the experiment. Effect of histamine on mucosal blood flow. Graded doses ofhistamine-0.25, 0.5, 1.0, and 2.0 J,Lg of base per kg per min were administered. In figure 2 antral and fundic aminopyrine clearance is correlated with acid secretion from the fundic pouch. Acid
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February ]971
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FIG. 3. Acid output (bars) from fundic pouch and aminopyrine clearance (circles) in antral and fundic pouches in response to histamine (2 Ilg of base per kg per min) infusion,
secretion and aminopyrine output of fundic pouches were obtained in two ways. In some experiments the acid instillations were continued throughout the experiment whereas in others, once the histamine was started, no further instillations were performed. There was no difference in either aminopyrine clearance or acid output in either group; the results are therefore combined. Aminopyrine clearance from the antrum increased with increasing doses of histamine, and had doubled with the highest dose used, a dose which elicits maximal acid output from the canine stomach. Fundic perfusion increased from a resting value of 0.72 ± 0.16 ml per min per g to 2.74 ± 0.31 ml per min per g. The antrum to corpus perfusion ratio dropped from 0.53: 1 to 0.27: 1. At maximal secretory rates, the rate of acid production was 8.10 ± 0.19 (SE) ~Eq per min per g of wet mucosa. When histamine was administered by continuous infusion for 2 hr and then discontinued (fig. 3), it is seen that a fall off in aminopyrine levels for both antrum and fundus occurred, so that resting values were obtained after 30 to 45 min. Secretion from the fundic pouch, on the other hand, had already fallen off after 15 min. Correlation of measured and extrapolated mucosal blood flow of resting fundus.
In the histamine-stimulated experiments, the fundic mucosal blood flow was plotted as a function of hydrogen ion output during steady states of acid secretion. There was a linear relationship between H + output and plasma aminopyrine clearance at all rates of secretion. By projecting the regression line in each dog it was possible to extrapolate mucosal blood flow at zero rate of secretion. Measured and extrapolated values showed good correlation in each dog (table 3); there were no significant differences between these two values (P > 0.02). The ratio between H + output in microequivalents per g of wet mucosa per min and plasma aminopyrine clearance in milliliters per g of wet mucosa per min during active secretion was 3.57 ± 0.27 (sn) : 1. Effects of isoproterenol and vasopressin. Isoproterenol, 1 ~g per kg per min, increased antral blood flow by 110% and resting fundic mucosal blood flow by 100% (fig. 4). Vasopressin decreased antral and resting fundic mucosal blood flow by 75 and 72%, respectively (fig. 5). 3. Comparison of aminopyrine clearance in nonsecreting fundic pouches obtained by direct measurement (acid instillations) and by extrapolation from values of the pouches at varying secretory rates a
TABLE
Dog
1 2 3 4 5 6 Mean
Measured
0.84 0.66 0.48 0.83 0.40 0,68
± ± ± ± ± ±
0.17 0.21 0.09 0.15 0,08 0.16
0.64 ± 0.23 b
Calculated
0.72 0.62 0.44 0.65 0.47 0.63
± ± ± ± ± ±
0.20 0.16 0.11 0.13 0.12 0.19
0.58 ± 0.21b
a Measured values are derived from aminopyrine clearance during the instillation of exogenous acid; calculated values are derived from the projected ami· nopyrine clearance at zero secretion, utilizing data from the histamine experiments in which ~aminopy rine clearance was plotted against hydrogen ion output. Results are given as means ± SD. b P > 0.02.
268
RUDICK ET AL.
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Alterations in ionic fluxes. In control experiments without aminopyrine, it was found that the net loss of hydrogen ions from each pouch remained constant over 5 hr. On the basis of unit surface area, the antrum was 15 times more permeable than the fundus, confirming our earlier report. 12
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With the continuous administration of aminopyrine, however, there was a greater loss of hydrogen ions from both pouches but particularly from the antral pouch with increasing time. The net loss of hydrogen ions was always accompanied by a net gain of sodium, although the sodium did not fully account for the loss of hydrogen ions. Discussion Our data lend further support to the validity of the aminopyrine clearance method of measuring gastric mucosal blood flow. The high correlation between calculated and projected aminopyrine clearance at zero secretion in the fundus attests to the validity of this method when using exogenous instead of secreted acid and is in agreement with the findings of others. 10 , 16, 17 Although it is impossible to compare calculated and projected aminopyrine clearances in the antrum there is no reason to believe that this method is any less valid for the antrum. The flow rates obtained (0.34 ml per min per g for antrum and 0.64 ml per min per g for fundus) are in general agreement with previous reported values. 18 Our figures are a little lower than those reported by Delaney and Grim. 3 However, these investigators studied the whole innervated stomach, whereas both our antral and fundic pouches were vagally denervated, and there is evidence to suggest that vagal denervation reduces both mucosap9 and totaPO gastric blood flow. Although there is an increasing body of evidence attesting to the validity of aminopyrine clearance as a measure of mucosal blood flow from the secreting fundus, we have to establish the validity for the antrum. We therefore investigated whether substances which produce primary alterations in blood flow would affect aminopyrine clearance from the antrum. Infusion of isoproterenol at a dose which has previously been shown to increase 'mucosal blood flow in the actively secreting fundus 6 increased both resting fundus mucosal blood flow and antral mucosal blood flow. Similarly, vasopressin which has
February 1971
MUCOSAL BLOOD FLOW
been shown to decrease mucosal blood flow in the actively secreting fundus 6 reduced aminopyrine clearance in both the unstimulated fundus and the antrum. While this technique allows a correlation of mucosal blood flow and function in the oxyntic gland area, WE: cannot draw any conclusions between blood flow and function in the antrum, since one of the principal functions of the antrum is the release of gastrin, which is inhibited at the pH of the solutions we used. Nevertheless, this represents a true state of affairs for the antrum in response to exogenous stimuli. When secretory tests are performed in separated fundic pouches, the antrum is usually bathed with acid gastric juice unless it is excluded or acid gastric juice is diverted by a cannula proximal to the antrum. Several criticisms, chiefly theoretical, have been raised against aminopyrine clearance as a measure of gastric mucosal blood flow. Our data answer some of these criticisms. One unanswered question has been whether aminopyrine clearance and acid secretion are linked by a common transport mechanism. The evidence against this is chiefly based on indirect evidence,21 but the clearance of aminopyrine in the resting nonsecreting fundus and in the antrum and its augmentation by vasodilators in the absence of acid secretion argue strongly against this. furthermore, the similarity of the recovery of instilled aminopyrine in the two different pouches while H + loss from the antrum exceeded that from the fundus discounts such a contention. Second, Moody7 suggested that aminopyrine may be cleared only from the portion of the capillary bed perfusing a region of actively secreting oxyntic cells. The movement of aminopyrine across the antrum indicates that the clearance is related to flow through the whole of the gastric mucosa rather than to that fraction which supplies the oxyntic cells. There are very few studies comparing antral and fundic blood flow. Rudick et al. 5 utilizing the ultrasonic flowmeter, compared blood flow but the technique only allowed a qualitative analysis of rel-
269
ative flow. M enguy2 reported that under resting conditions gastric blood flow was predominantly antral, and that antral perfusion decreased in response to histamine. Menguy's methodology has been criticized,3 however, and it is also possible that the technique used measured blood volume rather than blood flow. These two are not synonymous since alterations in venous resistance can affect blood flow and blood volume in diametrically opposite directions. The present data are in agreement with the findings of Delaney and Grim 3 and Rudick et al. 5 who found that histamine increased blood flow to both antrum and corpus. Histamine thus increased perfusion of both the parietal and nonparietal portions of gastric mucosa. Studies of gastric blood flow using aminopyrine clearance usually report the R value, which is approximately 40 for the actively secreting fundus. The R value, which in the actively secreting stomach is an index of the ratio of blood flow to secretory rate, is obtained from the formula, (gastric juice aminopyrine/plasma aminopyrine), and is also equal to (aminopyrine clearance/volume of gastric juice). With exogenous acid instillation, the R value would be dependent on blood flow, time, and the volume of fluid instilled (and recovered). In this study we have not presented data on this figure since the concentration of aminopyrine in the pouch contents could be altered simply by varying the amount of acid instilled into each pouch without affecting mucosal perfusion or clearance of aminopyrine. The maximal acid output of 8.10 ± 0.19 /lEq per min per g of wet mucosa in our study is similar to that reported by Cooke and Cooke 22 (7.60 /lEq per min per g). It is of interest that the ratio of H + output to plasma aminopyrine clearance during active secretion is 3.57: 1. This means that in the secretory state an increase in H + output of 1 /lEq per min per g of wet mucosa is accompanied by an increment in mucosal blood flow of 0.28 ml per min per g of wet mucosa. It is noteworthy that the continuous infusion of aminopyrine altered the permeability of both antral and fundic mucosa,
270
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RUDICK ET AL.
permitting an insorption of hydrogen ions and gain of sodium. Increased back diffusion of hydrogen has been demonstrated with salicylates. 23 However, Jacobson et al. 6 found that infusion of aminopyrine over a period of 1 hr did not affect the volume of acid secretion or hydrogen ion concentration of gastric juice from the actively secreting stomach. Nevertheless, despite the greater H+ loss in our experiments, plasma aminopyrine clearance remained constant. It has been suggested24 that the mucosal clearance of aminopyrine is dependent upon the proportion of the luminal surface acidified. Since we distended the pouches, it is likely that the instilled acid at least partially diffused into the gastric pits and tubules, thus increasing the surface area of the membrane for diffusion. The aminopyrine would theoretically come into contact with a larger surface area. Nevertheless, it is of interest that in the resting state it took 30 to 45 min for the aminopyrine clearance to reach a plateau. Either exogenous acid instillations increase mucosal blood flow, or it took this time for the aminopyrine to diffuse fully into the pits of the gastric mucosa. Certainly, the pH at the membrane surface rather than the pH of the bulk gastric contents determines the degree to which ionization of the aminopyrine occurs. The increased resting blood flow to both pouches noted with isoproterenol infusion demonstrates that an increase in gastric mucosal blood flow can occur without acid secretion in the absence of a secretory stimulant and is in agreement with other reports. 17, 25, 26 Thus, while primary changes in gastric secretion are usually accompanied by directionally corresponding changes in mucosal blood flow, the two can be dissociated. Although inhibitors of gastric secretion usually cause a reduction of blood flow concomitant with the decrease in secretion, it is not usually established whether the decrease in mucosal perfusion is primary or secondary. The demonstration that vasopressin reduces aminopyrine clearance from the unstimulated mucosa suggests that this method
may be utilized to determine whether the effects of gastric secretory inhibitors exert their effects by primary reductions in mucosal perfusion. Indeed, we have confirmed the study of Cowley et al. 27 that rapid supramaximal injections of pentagastrin probably cause their inhibitory effects in the dog by a primary reduction in fundic blood flow. 28 REFERENCES 1. Rudick J , Werther JL, Chapman ML, et al:
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February 1971
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MUCOSAL BLOOD FLOW
(Pyramidon) in man and methods for the estimation of aminopyrine and its metabolites in biological materiaL J Pharmacol Exp Ther 99:171180, 1960 Augur NA: Gastric mucosal blood flow following damage by ethanol, acetic acid, or aspirin. Gastroenterology 58:311-320, 1970 Cowley DJ, Code CF: Effects of secretory inhibitors on mucosal blood flow in non-secreting stomach of conscious dogs. Amer J Physiol 218: 270-274, 1970 Jacobson ED: The circulation of the stomach. Gastroenterology 48:85-109, 1965 Bell PRF, Battersby C: Effect of vagotomy on gastric mucosal blood flow. Gastroenterology 54:1032-1037, 1968 Rudick J, Guntheroth WG, Nyhus LM: Recent observations on gastric blood flow and acid secretion, Gastric Secretion ; Mechanisms and Control. Edited by TK Schnitka, Gilbert, and RC Harrison. New York, Pergamon Press, 1967, p 53-69 Jacobson ED: Clearances of the gastric mucosa. Gastroenterology 54:434-448, 1968
271
22 . Cooke HJ, Cooke AR: Correlation between the weight of gastric mucosa and maximal acid secretion. Digestion 1:209-212, 1968 23. Davenport HW : Gastric mucosal injury by fatty and acetyl-salicyclic acids. Gastroenterology 46: 245-253, 1964 24. Ellison RA, Nattie EE, Naitove A: Relation of gastric mucosal extraction and intraluminal appearance of aminopyrine. Amer J Physiol (in press) 25. Cutting WC, Dodds EC, Noble RL, et al: Pituitary control of alimentary blood flow and secretion. Proc Roy Soc [Bioi) 123:27-59, 1937 26. Nicoloff DM, Sosin H, Peter ET, et al: The effect of serotonin on gastric secretion. Amer J Dig Dis 8:267-272, 1963 27. Cowley DJ, Code CF, Fiasse R: Gastric mucosal blood flow during secretory inhibition by gastrin penta peptide and gastrone. Gastroenterology 56:659-665, 1969 28. Rudick J, Chapman, ML, Werther JL, et al: Influence of pentagastrin on mucosal blood flow in canine fundic and antral pouches. Clin Res 17:310, 1969
GASTROENTEROLOGY
Copyright
©
1971 by The Williams & Wilkins Co.
MUCOSAL BLOOD FLOW IN CANINE ANTRAL AND FUNDIC POUCHES JACK RUDICK, M.D., J. LAWRENCE WERTHER, M.D., MARK L. CHAPMAN, M.D., DAVID A. DREILING, M.D., AND HENRY D. JANOWITZ, M.D.
Department of Surgery and the Division of Gastroenterology. The Mount Sinai School of Medicine of the City University of New York, New York, New York
Aminopyrine clearance was used to compare mucosal blood flow in the resting and stimulated fundus and antrum. The intraluminal instillation of 0.16 N Hel provided a pH gradient allowing aminopyrine to move into the lumen. Resting mucosal perfusion was 0.64 ± 0.23 (so) ml per min per g of mucosa for the fundus and 0.34 ± 0.10 ml per min per g of wet mucosa for the antrum. Extrapolated mucosal blood flow of the resting fundus by plotting aminopyrine clearance against acid output during secretory stimulation correlated well with mucosal blood flow during acid instillations. Graded doses of histamine by intravenous infusion stimulated mucosal blood flow in the fundus parallel to acid secretion; a maximal dose (2 Ilg of base per kg per min) increased mucosal perfusion of the fundus 4-fold and doubled antral mucosal perfusion. Isoproterenol (1 Ilg per kg per min intravenously) increased mucosal blood flow to the resting fundus by 100% and to the antrum by 110%. Vasopressin (4 mU per kg per min intravenously) decreased resting fundic mucosal blood flow by 75% and antral blood flow by 80%. The present study lends further support to the validity of aminopyrine clearance as a measure of mucosal blood flow. It is further shown that: (1) aminopyrine clearance in the fundus and antrum is not dependent on the secretion of hydrogen ions and can be measured by using exogenous acid; (2) no redistribution of mucosal blood flow from antrum to corpus occurs with histamine stimulation; (3) primary alterations in mucosal blood flow can be induced in both pouches by vasoactive drugs without affecting acid secretion. Recent innovations in methodology 2-10 have revived interest in the measurement of gastric blood flow. Most studies, however, have concentrated either on the Received July 1, 1970. Accepted September 8, 1970. This work was presented in part to the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, April 18, 1969 and an abstract has appeared. 1 Address requests for reprints to: Dr. Jack Rudick, Department of Surgery, The Mount Sinai Hospital, 3 East l00th Street, New York, New York 10029. This work was supported by United States Public Health Service Grant AM-11580 from the National Institutes of Arthritis and Metabolic Diseases. Dr. Rudick is a recipient of Career Scientist Award 1-541 from the New York Health Research Council.
whole stomach or on the oxyntic gland area. Within certain limitations, the evidence points to a general relationship between gastric blood flow and acid secretion. Aminopyrine clearance from the gastric mucosa has been confirmed as a reliable method for the measurement of mucosal blood flow. 6 - 1o This method is dependent upon a pH gradient for aminopyrine to move into gastric juice. Since a pH in the gastric lumen less than the pK of aminopyrine (pK 5) is required, little information exists on mucosal blood flow in the antrum (a non-acid secreting portion of the stomach) or on the unstimulated corpus. In this study we have utilized exogenous 263
RUDICK ET AL.
264
acid to provide a pH gradient between plasma and gastric lumen. Aminopyrine clearance has thus been measured from the antrum and compared to the fundus. We have also attempted to determine whether alterations in blood flow are associated with fluxes of ions across the mucosa.
Methods Eight dogs of both sexes ranging in weight from 18 to 28 kg were used . Four dogs were prepared with vagally denervated antral and vagally denervated fundic pouches, 2 dogs with antral pouches only, and 2 dogs with fundic pouches alone. Pure antral pouches were obtained by delineating the antrum-corpus junction colorimetrically with 1 % toluidine blue intravenously.ll A 2-cm rim of tissue at the intermediate zone between antrum and corpus was excised to ensure a pure antral pouch preparation . This was subsequently confirmed by (a) histological examination of the intermediate zone and (b) monitoring antral pH during the intravenous administration of histamine (2 ILg of base per kg per min). Each pouch was equipped with a nylon-coated stainless steel gastric cannula which was exteriorized in the midline to assist complete emptying of the pouch when the animals stood upright during the test. The animals remained in good health and maintained stable weights during the period of study. At least 4 weeks elapsed before experiments were started. Experiments were conducted after the dogs had been deprived of food but not water of 18 hr prior to each observation. Each dog was studied no more than once a week. The animals were placed in a modified Pavlov frame and 0.9% saline was infused intravenously at a constant rate (0.6 ml per min). A loading dose of aminopyrine (20 mg per kg) was injected over 10 min, and was followed by a maintenance dose of 5 mg per kg hr throughout each experiment. The intralumenal instillation of 0.16 N HCI provided a pH gradient allowing aminopyrine to migrate into the lumen of each pouch. The volumes of acid solutions instilled were determined individually for each pouch. One end of a polyethelene tube was attached to an adaptor screwed on tightly into the gastric cannula and the other end was attached to a three-way stopcock. Antral pouches were filled to a hydrostatic pressure of 15 cm, and fundic pouches to a hydrostatic pressure of 6 cm. Capacities of the antral pouches ranged from 18 to 35 ml and the fundic pouches from
Vol . 60, No . 2
60 to 100 ml. During determinations of the pouch volumes and in subsequent experiments it was demonstrated that no leak was present. At the beginning of each experiment, each pouch was washed out with 0.16 N HCI warmed to 37 C, and the pouch was drained as completely as possible. The predetermined volume of acid with a nonabsorbable marker (4 mg per 100 ml of phenol red) was then introduced into the pouch and, after mixing, a 3-ml aliquot was removed to determine the residual volume. The acid solution was allowed to remain in the pouch for 15 min and then emptied by gravity drainage and gentle massage over the pouch area; a fresh solution was then introduced. Residual volumes were determined in the manner previously described. 12 Blood samples were obtained from a peripheral leg vein every 30 min. The volume of fluid recovered from each pouch was determined to the nearest 0.1 ml. Specimens were analyzed individually for volume, acidity, sodium, potassium, chloride, phenol red, osmolality, and aminopyrine concentration. Acidity was measured by titration to end point pH 7.0 with a pH-stat titrator (Radiometer TIT1c). Sodium, potassium, and chloride were determined with a Technicon AutoAnalyzer and osmolality with an advanced osmometer. Phenol red determinations were performed according to the method of Hunt. IS Aminopyrine clearance was determined by the method of Shore et al. 1. as adapted by Jacobson et al. 6 to measure mucosal plasma flow . Pouch contents and plasma were analyzed individually for aminopyrine concentration by the method of Brodie and Axelrod. 1 ' Mucosal blood flow in both pouches was measured: (a) in the resting state; (b) in response to graded doses of histamine, 0.25 to 2.0 ILg of base per kg per min; (c) in response to isoproterenol (Isuprel, Winthrop-Stearns, Inc.), 1 ILg per kg per min, and to Vasopressin (Parke, Davis and Co.), 4 m U per kg per min. In order to allow comparison with other studies (utilizing a variety of techniques), all of the pouches were excised after termination of the studies. The pouches were excised with the animals under general anesthesia and the gross fat was trimmed off. The mucosa was stripped from the underlying submucosa, gently blotted free of gross moisture, and weighed while wet. Mucosa of antrum was 33.0 ± 2.5% (so) of the pouch weight, while the mucosa of the fundus was 47.8 ± 4.2 % of the pouch weight. The weight of the mucosa of the pouches did not correlate with body weight because the size of the pouch was dependent not only on the size
of the dog (and stomach) but also on technical considerations, so that a larger animal may have had a smaller pouch. The results are therefore expressed as a function of mucosal weight rather than weight of the animal. Plasma clearance of aminopyrine was derived from the formula: GV c=---P X t X w
where C is plasma aminopyrine clearance in milliliters per min per g of wet mucosa; G is concentration of aminopyrine in gastric contents; V is volume of gastric contents at the end of each instillation period; P is plasma aminopyrine concentration; t is time in minutes; and w is weight of wet mucosa. Secretion of acid in the histamine experiments was expressed as microequivalents per g of wet mucosa per min. The surface area of the mucosa was measured by planimetry, enabling ionic movements to be determined per unit area (square centimeters). During the intravenous administration of aminopyrine in 2 of the dogs with double pouches, either 0.16 N HCI or isotonic mannitol (5.82 per 100 ml at pH 6.8 was instilled into each pouch; recovery of aminopyrine was compared when the pouches were acidified or neutral. In separate experiments in these 2 animals, 50 mg of aminopyrine dissolved in 0.16 N HCI were introduced into each pouch and the aminopyrine recovered in the pouch contents was determined at the end of 15 min. During the latter experiments, no aminopyrine was administered intravenously.
Results Recovery of aminopyrine. Recovery of aminopyrine from acidified antral and fun1. Aminopyrine recovery from antral and fundic pouches when acid (0 .16 N HCl) or neutral (mannitol) solutions were instilled into each pouch"
TABLE
Aminopyrine recovered Solution instilled
n'
I
~--------.----------
Antrum
Fundus
mg/I5 min
0.16N HCL ... Mannitol ...
265
MUCOSAL BLOOD FLOW
February 1971
35 1.12 ± 0.28·13.12 ± 0.52 33 0.19 ± 0.0510.52 ± 0.18
• Results are given as mean ± b n, no. of observations.
SD.
TABLE
2. Plasma aminopyrine clearance in resting
antral and fundic pouches' Dog
Antrum
Fundus
mllmin/g wet mucosa
1 2 3 4 5 6 7 8
Mean
0.22 0.46 0 . 21 0 . 39 0 . 43 0 . 31
± ± ± ± ± ±
0.08 0 .06 0.07 0.13 0.10 0.03
0.34 ± 0.10
• Results are given as mean ±
0.84 0 . 66 0.48 0.83 0 .50 0.58
± ± ± ± ± ±
0.17 0.21 0 .09 0 . 15 0 .08 0.16
0.64 ± 0.23
SD.
dic pouches in 2 dogs is seen in table 1. When isotonic mannitol at pH 6.8 instead of acid was instilled it resulted in a much reduced recovery of aminopyrine from each pouch, despite the maintenance of a constant plasma aminopyrine level. When 50 mg of aminopyrine dissolved in 0.16 N Hel were introduced into each pouch in the absence of circulating aminopyrine, recovery was 96.7 ± 3.6% (SE) for antral pouches and 97.8 ± 2.9% for fundic pouches. No significant absorption of aminopyrine was detected from the lumen of acidified pouches. In contrast, aminopyrine dissolved in mannitol at pH 6.8 almost completely disappeared from each pouch in 5 to 10 min. Resting mucosal blood flow in unstimulated antral and fundic pouches. Mean perfusion was 0.34 ± 0.10 (mean ± so) ml per min per g of wet mucosa for the antrum and 0.64 ± 0.23 ml per min per g of wet mucosa for the corpus. Individual values of each dog are seen in table 2. These figures are derived from resting values when aminopyrine clearance had stabilized. The perfusion ratio of antrum to corpus is thus 0.53: 1. It was found that it usually took about 45 to 60 min for the concentration of aminopyrine to reach a plateau in the pouch contents. Mean results from six experiments in 3 dogs are illustrated in figure 1. Antral aminopyrine clearance of 0.32 ± 0.06 ml per min per g of wet mucosa was 22% lower than a simi-
266
RUDICK E T AL.
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lar instillation period after a plateau had been reached. Similarly, corpus aminopyrine clearance of 0.58 ± 0.08 ml per min per g of wet mucosa was 27% less than when perfusion had stabilized. To test further the validity of the clearance method, the length of instillation periods was varied. Once a plateau had been reached there was no difference in the perfusion per unit time when the instillation periods were varied from 15 to 30 or 60 min. It is also evident from figure 1 that there was no decline in aminopyrine clear-
ance with the passage of tIme, provided that plasma aminopyrine concentration remained stable. This is of interest since, with the passage of time there was frequently a greater loss of hydrogen ions from the antral pouches than at the beginning of the experiment. Effect of histamine on mucosal blood flow. Graded doses ofhistamine-0.25, 0.5, 1.0, and 2.0 J,Lg of base per kg per min were administered. In figure 2 antral and fundic aminopyrine clearance is correlated with acid secretion from the fundic pouch. Acid
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February ]971
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FIG. 3. Acid output (bars) from fundic pouch and aminopyrine clearance (circles) in antral and fundic pouches in response to histamine (2 Ilg of base per kg per min) infusion,
secretion and aminopyrine output of fundic pouches were obtained in two ways. In some experiments the acid instillations were continued throughout the experiment whereas in others, once the histamine was started, no further instillations were performed. There was no difference in either aminopyrine clearance or acid output in either group; the results are therefore combined. Aminopyrine clearance from the antrum increased with increasing doses of histamine, and had doubled with the highest dose used, a dose which elicits maximal acid output from the canine stomach. Fundic perfusion increased from a resting value of 0.72 ± 0.16 ml per min per g to 2.74 ± 0.31 ml per min per g. The antrum to corpus perfusion ratio dropped from 0.53: 1 to 0.27: 1. At maximal secretory rates, the rate of acid production was 8.10 ± 0.19 (SE) ~Eq per min per g of wet mucosa. When histamine was administered by continuous infusion for 2 hr and then discontinued (fig. 3), it is seen that a fall off in aminopyrine levels for both antrum and fundus occurred, so that resting values were obtained after 30 to 45 min. Secretion from the fundic pouch, on the other hand, had already fallen off after 15 min. Correlation of measured and extrapolated mucosal blood flow of resting fundus.
In the histamine-stimulated experiments, the fundic mucosal blood flow was plotted as a function of hydrogen ion output during steady states of acid secretion. There was a linear relationship between H + output and plasma aminopyrine clearance at all rates of secretion. By projecting the regression line in each dog it was possible to extrapolate mucosal blood flow at zero rate of secretion. Measured and extrapolated values showed good correlation in each dog (table 3); there were no significant differences between these two values (P > 0.02). The ratio between H + output in microequivalents per g of wet mucosa per min and plasma aminopyrine clearance in milliliters per g of wet mucosa per min during active secretion was 3.57 ± 0.27 (sn) : 1. Effects of isoproterenol and vasopressin. Isoproterenol, 1 ~g per kg per min, increased antral blood flow by 110% and resting fundic mucosal blood flow by 100% (fig. 4). Vasopressin decreased antral and resting fundic mucosal blood flow by 75 and 72%, respectively (fig. 5). 3. Comparison of aminopyrine clearance in nonsecreting fundic pouches obtained by direct measurement (acid instillations) and by extrapolation from values of the pouches at varying secretory rates a
TABLE
Dog
1 2 3 4 5 6 Mean
Measured
0.84 0.66 0.48 0.83 0.40 0,68
± ± ± ± ± ±
0.17 0.21 0.09 0.15 0,08 0.16
0.64 ± 0.23 b
Calculated
0.72 0.62 0.44 0.65 0.47 0.63
± ± ± ± ± ±
0.20 0.16 0.11 0.13 0.12 0.19
0.58 ± 0.21b
a Measured values are derived from aminopyrine clearance during the instillation of exogenous acid; calculated values are derived from the projected ami· nopyrine clearance at zero secretion, utilizing data from the histamine experiments in which ~aminopy rine clearance was plotted against hydrogen ion output. Results are given as means ± SD. b P > 0.02.
268
RUDICK ET AL.
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Alterations in ionic fluxes. In control experiments without aminopyrine, it was found that the net loss of hydrogen ions from each pouch remained constant over 5 hr. On the basis of unit surface area, the antrum was 15 times more permeable than the fundus, confirming our earlier report. 12
Vol . 60, No.2
With the continuous administration of aminopyrine, however, there was a greater loss of hydrogen ions from both pouches but particularly from the antral pouch with increasing time. The net loss of hydrogen ions was always accompanied by a net gain of sodium, although the sodium did not fully account for the loss of hydrogen ions. Discussion Our data lend further support to the validity of the aminopyrine clearance method of measuring gastric mucosal blood flow. The high correlation between calculated and projected aminopyrine clearance at zero secretion in the fundus attests to the validity of this method when using exogenous instead of secreted acid and is in agreement with the findings of others. 10 , 16, 17 Although it is impossible to compare calculated and projected aminopyrine clearances in the antrum there is no reason to believe that this method is any less valid for the antrum. The flow rates obtained (0.34 ml per min per g for antrum and 0.64 ml per min per g for fundus) are in general agreement with previous reported values. 18 Our figures are a little lower than those reported by Delaney and Grim. 3 However, these investigators studied the whole innervated stomach, whereas both our antral and fundic pouches were vagally denervated, and there is evidence to suggest that vagal denervation reduces both mucosap9 and totaPO gastric blood flow. Although there is an increasing body of evidence attesting to the validity of aminopyrine clearance as a measure of mucosal blood flow from the secreting fundus, we have to establish the validity for the antrum. We therefore investigated whether substances which produce primary alterations in blood flow would affect aminopyrine clearance from the antrum. Infusion of isoproterenol at a dose which has previously been shown to increase 'mucosal blood flow in the actively secreting fundus 6 increased both resting fundus mucosal blood flow and antral mucosal blood flow. Similarly, vasopressin which has
February 1971
MUCOSAL BLOOD FLOW
been shown to decrease mucosal blood flow in the actively secreting fundus 6 reduced aminopyrine clearance in both the unstimulated fundus and the antrum. While this technique allows a correlation of mucosal blood flow and function in the oxyntic gland area, WE: cannot draw any conclusions between blood flow and function in the antrum, since one of the principal functions of the antrum is the release of gastrin, which is inhibited at the pH of the solutions we used. Nevertheless, this represents a true state of affairs for the antrum in response to exogenous stimuli. When secretory tests are performed in separated fundic pouches, the antrum is usually bathed with acid gastric juice unless it is excluded or acid gastric juice is diverted by a cannula proximal to the antrum. Several criticisms, chiefly theoretical, have been raised against aminopyrine clearance as a measure of gastric mucosal blood flow. Our data answer some of these criticisms. One unanswered question has been whether aminopyrine clearance and acid secretion are linked by a common transport mechanism. The evidence against this is chiefly based on indirect evidence,21 but the clearance of aminopyrine in the resting nonsecreting fundus and in the antrum and its augmentation by vasodilators in the absence of acid secretion argue strongly against this. furthermore, the similarity of the recovery of instilled aminopyrine in the two different pouches while H + loss from the antrum exceeded that from the fundus discounts such a contention. Second, Moody7 suggested that aminopyrine may be cleared only from the portion of the capillary bed perfusing a region of actively secreting oxyntic cells. The movement of aminopyrine across the antrum indicates that the clearance is related to flow through the whole of the gastric mucosa rather than to that fraction which supplies the oxyntic cells. There are very few studies comparing antral and fundic blood flow. Rudick et al. 5 utilizing the ultrasonic flowmeter, compared blood flow but the technique only allowed a qualitative analysis of rel-
269
ative flow. M enguy2 reported that under resting conditions gastric blood flow was predominantly antral, and that antral perfusion decreased in response to histamine. Menguy's methodology has been criticized,3 however, and it is also possible that the technique used measured blood volume rather than blood flow. These two are not synonymous since alterations in venous resistance can affect blood flow and blood volume in diametrically opposite directions. The present data are in agreement with the findings of Delaney and Grim 3 and Rudick et al. 5 who found that histamine increased blood flow to both antrum and corpus. Histamine thus increased perfusion of both the parietal and nonparietal portions of gastric mucosa. Studies of gastric blood flow using aminopyrine clearance usually report the R value, which is approximately 40 for the actively secreting fundus. The R value, which in the actively secreting stomach is an index of the ratio of blood flow to secretory rate, is obtained from the formula, (gastric juice aminopyrine/plasma aminopyrine), and is also equal to (aminopyrine clearance/volume of gastric juice). With exogenous acid instillation, the R value would be dependent on blood flow, time, and the volume of fluid instilled (and recovered). In this study we have not presented data on this figure since the concentration of aminopyrine in the pouch contents could be altered simply by varying the amount of acid instilled into each pouch without affecting mucosal perfusion or clearance of aminopyrine. The maximal acid output of 8.10 ± 0.19 /lEq per min per g of wet mucosa in our study is similar to that reported by Cooke and Cooke 22 (7.60 /lEq per min per g). It is of interest that the ratio of H + output to plasma aminopyrine clearance during active secretion is 3.57: 1. This means that in the secretory state an increase in H + output of 1 /lEq per min per g of wet mucosa is accompanied by an increment in mucosal blood flow of 0.28 ml per min per g of wet mucosa. It is noteworthy that the continuous infusion of aminopyrine altered the permeability of both antral and fundic mucosa,
270
Vol. 60, No.2
RUDICK ET AL.
permitting an insorption of hydrogen ions and gain of sodium. Increased back diffusion of hydrogen has been demonstrated with salicylates. 23 However, Jacobson et al. 6 found that infusion of aminopyrine over a period of 1 hr did not affect the volume of acid secretion or hydrogen ion concentration of gastric juice from the actively secreting stomach. Nevertheless, despite the greater H+ loss in our experiments, plasma aminopyrine clearance remained constant. It has been suggested24 that the mucosal clearance of aminopyrine is dependent upon the proportion of the luminal surface acidified. Since we distended the pouches, it is likely that the instilled acid at least partially diffused into the gastric pits and tubules, thus increasing the surface area of the membrane for diffusion. The aminopyrine would theoretically come into contact with a larger surface area. Nevertheless, it is of interest that in the resting state it took 30 to 45 min for the aminopyrine clearance to reach a plateau. Either exogenous acid instillations increase mucosal blood flow, or it took this time for the aminopyrine to diffuse fully into the pits of the gastric mucosa. Certainly, the pH at the membrane surface rather than the pH of the bulk gastric contents determines the degree to which ionization of the aminopyrine occurs. The increased resting blood flow to both pouches noted with isoproterenol infusion demonstrates that an increase in gastric mucosal blood flow can occur without acid secretion in the absence of a secretory stimulant and is in agreement with other reports. 17, 25, 26 Thus, while primary changes in gastric secretion are usually accompanied by directionally corresponding changes in mucosal blood flow, the two can be dissociated. Although inhibitors of gastric secretion usually cause a reduction of blood flow concomitant with the decrease in secretion, it is not usually established whether the decrease in mucosal perfusion is primary or secondary. The demonstration that vasopressin reduces aminopyrine clearance from the unstimulated mucosa suggests that this method
may be utilized to determine whether the effects of gastric secretory inhibitors exert their effects by primary reductions in mucosal perfusion. Indeed, we have confirmed the study of Cowley et al. 27 that rapid supramaximal injections of pentagastrin probably cause their inhibitory effects in the dog by a primary reduction in fundic blood flow. 28 REFERENCES 1. Rudick J , Werther JL, Chapman ML, et al:
2. 3. 4. 5.
6.
7. 8. 9. 10.
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12.
13.
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