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Removal of gastrin by various organs in dogs
00165985/78/0074-ooo1$02.09/0 GMTEO~TMKILOIX14:32-33,1978 Copyright 0 1978 by the AmericanGastroenterological Association
Vol.74,No.1
Printed in USA.
REMOVAL OF’GASTRIN BY VARIOUS ORGANS IN DOGS ULRICHT. STRUNZ,JOHNH. WALSH,ANDMORTONI. GROSSMAN Veterans Administration California
Wadsworth Hospital Center and UCLA School of Medicine, Los Angeles,
In 6 anesthetized dogs receiving 400 pmoles kg’ hr-’ of natural human little gastrin intravenously, blood samples for radioimmunoassay of gastrin were taken repeatedly from a carotid artery, jugular vein, femoral vein, renal vein, and mesenteric vein. The calculated removal of gastrin in a single passage through these four vascular beds ranged from 21 to 30% and did not differ significantly among them. In 6 additional dogs, the carotid arterial to hepatic venous removal was 40%. We conclude that the percentage removal of gastrin is about the same in all of the major vascular beds of the body. A number of studies (reviewed in Reference 1) have shown that the venous blood from organs such as the kidney, intestine, liver, or stomach has a lower concentration of immunoassayable gastrin than arterial blood simultaneously sampled. Such findings led to the notion that these organs play a special role in the metabolism of gastrin. The present study showed that during continuous intravenous infusion of little gastrin which produced a plateau of blood gastrin concentration, the arteriovenous differences across the kidney, intestine, and liver were no greater than across the head or a rear leg. This indicates that gastrin removal is a general property of most and perhaps all capillary beds.
to change during the 30 min of sampling, so for each sampling site in each dog the mean gastrin concentration was calculated. Percentage removal for each site and each dog was calculated by subtracting mean venous concentration from mean arterial concentration, dividing by mean arterial concentration, and multiplying by 100. These individual-dog percentage removals were used to calculate the between-dogs means and standard errors reported here. The significance of differences between means was determined by use of Student’s t-test, paired or unpaired as appropriate.
Results The mean arterial gastrin concentration was 441 * 33 fmoles ml-’ and this was significantly
(paired t-test,
P < 0.05) greater than the concentration at each of the
Methods
venous sampling sites. The mean percentage removal (table 1) by the head (jugular vein), intestine (mesenteric vein), kidney (renal vein), and rear leg (femoral vein), ranged from 21.0 to 30.4% without significant differences between sites (paired t-tests, P > 0.05). Similarly the arterial to hepatic vein removal, 40.1% did not differ significantly from that of theiother sites (unpaired t-tests, P > 0.05) (see table 1).
Sixdogs (17 to 22 kg) were anesthetized with sodium
pentothal, 25 mg kgl, and then placed on inhalation anesthesia (Penthrane, Abbott Laboratories, North Chicago, 111.)for the remainder of the study. Through a catheter in the right jugular vein natural human little gastrin (kind gift of R. A. Gregory and H. J. Tracy) was infused at a rate of 400 pmoles kg’ ~-IT’during the entire study. Beginning 60 min after starting the infusion of gastrin, blood was drawn at 5-min intervals for 30 min from the following vessels which had been exposed by dissection: right carotid artery, left jugular vein, right or left femoral vein, a mesenteric vein, and left renal vein. In another 6 dogs (18 to 22 kg) a similar study was done but blood was sampled only from the right carotid artery and the right hepatic vein. Blood was allowed to clot in tubes placed in an ice-water bath. Serum was separated from the blood samples within 1 hr of collection and then stored at -20°C until gastrin was determined by radioimmunoassay using antibody 1296 as described before.* Gastrin concentrations showed no tendency
Clearance rate and volume of distribution was calculated as previously described.3 Clearance rate was 15.1 ml kg1 mine1 when calculated from plateau arterial gastrin concentrations. Volume of distribution was 6.5% of body weight, assuming that the disappearance halftime was 3 min. These values were 20 to 30% higher when venous gastrin concentrations were used for the calculations.
Discussion In the present study, infusions of gastrin were performed in order to facilitate the measurement of arterial and venous concentrations of little gastrin. Basal and food-stimulated gastrin in the dog circulates in multiple molecular forms, and the accurate measurement of each form in physiological concentrations is difficult with current methodology. In previous studies it was shown that the mechanisms responsible for clearance of little gastrin are not influenced by the dose of gastrin
Received May 10, 1977. Accepted August 2, 1977. Address requests for reprints to: Dr. M. I. Grossman, Veterans Administration Wadsworth Hospital Center, Building 115, Room 115, Los Angeles, California 90073. This study was supported by Veterans Administration Senior Medical Investigatorship (M.I. Grossman), Grant 17328 to CURE (Center for Ulcer Research and Education), and Grant 17294 from the National Institute of Arthritis, Metabolism and Digestive Diseases. 32
January TABLE
1978
1.Percentage
Organ
Leg
Intestine Head Kidney Liver + intestine
REMOVAL
OF GASTRIN
removal (mean k SE for 6 dogs) of little gastrin bv various organs
Vein Femoral Mesenteric Jugular Renal Hepatic
Percentageremoval 21.0 k 26.2 + 29.2 + 30.4 k 40.1 +
4.8 4.3 4.6 3.3 4.8
infused up to doses of 400 pmoles kg’ h~‘.~ Also it has been shown that gastrin is not taken up and subsequently released from the capillary beds because serum gastrin concentrations in venous blood decreased exponentially over time as soon as gastrin infusions are stopped and do not exhibit an initial rise. In the present study, the concentration of gastrin at each sampling site did not vary during the test, indicating that a steady state had been reached as would be expected after a 60-min infusion of a hormone with a half-life of about 3 min.’ The values obtained for clearance and volume of distribution differed from those obtained previously in unanesthetized dogs which received porcine G-17 infusions.3 The less rapid clearance rate in the present study could be explained by two major factors other than anesthesia. First, they were calculated from arterial blood concentrations which were consistently higher than venous concentrations. Second, the samples were handled in a way which minimized loss of immunoreactive gastrin during storage, presumably attributable to the action of enzymes present in serum. The clearance rate of 15.1 ml kg’ min-’ was equivalent to approximately 27% of estimated resting cardiac plasma output in dogs (55 ml kg’ min-‘). The percentage removal of gastrin did not differ significantly in the various vascular beds sampled, indicating that the mechanism of removal is present in most or all tissues rather than only in certain ones. The highest percentage of removal was seen in the
BY
VARIOUS
33
ORGANS
hepatic vein, 40%. This value includes removal by both the intestine and the liver. Although this intestinal plus hepatic removal of 40% was not statistically significantly different from the 26% for intestine alone, it seems likely that the liver does in fact remove gastrin at a rate comparable to that of other organs. If one assumes that the difference between intestinal removal alone (26%) and intestinal plus hepatic removal (40%) is a true difference, and one further assumes that onethird of hepatic flow is arterial, the calculated removal by the liver itself would be 23%, similar to that of the other organs. The hepatic contribution to gastrin removal could probably have been more clearly demonstrated if mesenteric and hepatic venous blood had been collected in the same animals. ‘Ihe difficulty in exposing the hepatic veins made this impractical. The present study did not identify the exact site or mechanism of gastrin removal within the various organs. Circulating proteases are unlikely to contribute to the observed differences in arterial and venous gastrin concentrations because gastrin added to whole blood is not significantly inactivated after standing for up to 30 min at 37°C (unpublished data). The time blood remains in the capillary beds is at least an order of magnitude shorter than 30 min. It is possible that gastrin is degraded by proteases in the vascular endothelium or elsewhere in the tissues. However, it is clear from the present experiments that gastrin is removed at multiple sites throughout the body. REFERENCES 1. Walsh JH, Grossman MI: Gastrin. N Engl J Med 292:1324-1334, 1377-1384, 1975 2. Walsh JH: Radioimmunoassay of gastrin. In Nuclear Medicine in Vitro. Edited by B Rothfield. Philadelphia, JB Lippincott, 1974, p 231-248 3. Walsh JH, Debas HT, Grossman MI: Pure human big gastrin; immunochemical properties, disappearance half time, and acid stimulating action in dogs. J Clin Invest 54477~465,1974
Vol.74,No.1
Printed in USA.
REMOVAL OF’GASTRIN BY VARIOUS ORGANS IN DOGS ULRICHT. STRUNZ,JOHNH. WALSH,ANDMORTONI. GROSSMAN Veterans Administration California
Wadsworth Hospital Center and UCLA School of Medicine, Los Angeles,
In 6 anesthetized dogs receiving 400 pmoles kg’ hr-’ of natural human little gastrin intravenously, blood samples for radioimmunoassay of gastrin were taken repeatedly from a carotid artery, jugular vein, femoral vein, renal vein, and mesenteric vein. The calculated removal of gastrin in a single passage through these four vascular beds ranged from 21 to 30% and did not differ significantly among them. In 6 additional dogs, the carotid arterial to hepatic venous removal was 40%. We conclude that the percentage removal of gastrin is about the same in all of the major vascular beds of the body. A number of studies (reviewed in Reference 1) have shown that the venous blood from organs such as the kidney, intestine, liver, or stomach has a lower concentration of immunoassayable gastrin than arterial blood simultaneously sampled. Such findings led to the notion that these organs play a special role in the metabolism of gastrin. The present study showed that during continuous intravenous infusion of little gastrin which produced a plateau of blood gastrin concentration, the arteriovenous differences across the kidney, intestine, and liver were no greater than across the head or a rear leg. This indicates that gastrin removal is a general property of most and perhaps all capillary beds.
to change during the 30 min of sampling, so for each sampling site in each dog the mean gastrin concentration was calculated. Percentage removal for each site and each dog was calculated by subtracting mean venous concentration from mean arterial concentration, dividing by mean arterial concentration, and multiplying by 100. These individual-dog percentage removals were used to calculate the between-dogs means and standard errors reported here. The significance of differences between means was determined by use of Student’s t-test, paired or unpaired as appropriate.
Results The mean arterial gastrin concentration was 441 * 33 fmoles ml-’ and this was significantly
(paired t-test,
P < 0.05) greater than the concentration at each of the
Methods
venous sampling sites. The mean percentage removal (table 1) by the head (jugular vein), intestine (mesenteric vein), kidney (renal vein), and rear leg (femoral vein), ranged from 21.0 to 30.4% without significant differences between sites (paired t-tests, P > 0.05). Similarly the arterial to hepatic vein removal, 40.1% did not differ significantly from that of theiother sites (unpaired t-tests, P > 0.05) (see table 1).
Sixdogs (17 to 22 kg) were anesthetized with sodium
pentothal, 25 mg kgl, and then placed on inhalation anesthesia (Penthrane, Abbott Laboratories, North Chicago, 111.)for the remainder of the study. Through a catheter in the right jugular vein natural human little gastrin (kind gift of R. A. Gregory and H. J. Tracy) was infused at a rate of 400 pmoles kg’ ~-IT’during the entire study. Beginning 60 min after starting the infusion of gastrin, blood was drawn at 5-min intervals for 30 min from the following vessels which had been exposed by dissection: right carotid artery, left jugular vein, right or left femoral vein, a mesenteric vein, and left renal vein. In another 6 dogs (18 to 22 kg) a similar study was done but blood was sampled only from the right carotid artery and the right hepatic vein. Blood was allowed to clot in tubes placed in an ice-water bath. Serum was separated from the blood samples within 1 hr of collection and then stored at -20°C until gastrin was determined by radioimmunoassay using antibody 1296 as described before.* Gastrin concentrations showed no tendency
Clearance rate and volume of distribution was calculated as previously described.3 Clearance rate was 15.1 ml kg1 mine1 when calculated from plateau arterial gastrin concentrations. Volume of distribution was 6.5% of body weight, assuming that the disappearance halftime was 3 min. These values were 20 to 30% higher when venous gastrin concentrations were used for the calculations.
Discussion In the present study, infusions of gastrin were performed in order to facilitate the measurement of arterial and venous concentrations of little gastrin. Basal and food-stimulated gastrin in the dog circulates in multiple molecular forms, and the accurate measurement of each form in physiological concentrations is difficult with current methodology. In previous studies it was shown that the mechanisms responsible for clearance of little gastrin are not influenced by the dose of gastrin
Received May 10, 1977. Accepted August 2, 1977. Address requests for reprints to: Dr. M. I. Grossman, Veterans Administration Wadsworth Hospital Center, Building 115, Room 115, Los Angeles, California 90073. This study was supported by Veterans Administration Senior Medical Investigatorship (M.I. Grossman), Grant 17328 to CURE (Center for Ulcer Research and Education), and Grant 17294 from the National Institute of Arthritis, Metabolism and Digestive Diseases. 32
January TABLE
1978
1.Percentage
Organ
Leg
Intestine Head Kidney Liver + intestine
REMOVAL
OF GASTRIN
removal (mean k SE for 6 dogs) of little gastrin bv various organs
Vein Femoral Mesenteric Jugular Renal Hepatic
Percentageremoval 21.0 k 26.2 + 29.2 + 30.4 k 40.1 +
4.8 4.3 4.6 3.3 4.8
infused up to doses of 400 pmoles kg’ h~‘.~ Also it has been shown that gastrin is not taken up and subsequently released from the capillary beds because serum gastrin concentrations in venous blood decreased exponentially over time as soon as gastrin infusions are stopped and do not exhibit an initial rise. In the present study, the concentration of gastrin at each sampling site did not vary during the test, indicating that a steady state had been reached as would be expected after a 60-min infusion of a hormone with a half-life of about 3 min.’ The values obtained for clearance and volume of distribution differed from those obtained previously in unanesthetized dogs which received porcine G-17 infusions.3 The less rapid clearance rate in the present study could be explained by two major factors other than anesthesia. First, they were calculated from arterial blood concentrations which were consistently higher than venous concentrations. Second, the samples were handled in a way which minimized loss of immunoreactive gastrin during storage, presumably attributable to the action of enzymes present in serum. The clearance rate of 15.1 ml kg’ min-’ was equivalent to approximately 27% of estimated resting cardiac plasma output in dogs (55 ml kg’ min-‘). The percentage removal of gastrin did not differ significantly in the various vascular beds sampled, indicating that the mechanism of removal is present in most or all tissues rather than only in certain ones. The highest percentage of removal was seen in the
BY
VARIOUS
33
ORGANS
hepatic vein, 40%. This value includes removal by both the intestine and the liver. Although this intestinal plus hepatic removal of 40% was not statistically significantly different from the 26% for intestine alone, it seems likely that the liver does in fact remove gastrin at a rate comparable to that of other organs. If one assumes that the difference between intestinal removal alone (26%) and intestinal plus hepatic removal (40%) is a true difference, and one further assumes that onethird of hepatic flow is arterial, the calculated removal by the liver itself would be 23%, similar to that of the other organs. The hepatic contribution to gastrin removal could probably have been more clearly demonstrated if mesenteric and hepatic venous blood had been collected in the same animals. ‘Ihe difficulty in exposing the hepatic veins made this impractical. The present study did not identify the exact site or mechanism of gastrin removal within the various organs. Circulating proteases are unlikely to contribute to the observed differences in arterial and venous gastrin concentrations because gastrin added to whole blood is not significantly inactivated after standing for up to 30 min at 37°C (unpublished data). The time blood remains in the capillary beds is at least an order of magnitude shorter than 30 min. It is possible that gastrin is degraded by proteases in the vascular endothelium or elsewhere in the tissues. However, it is clear from the present experiments that gastrin is removed at multiple sites throughout the body. REFERENCES 1. Walsh JH, Grossman MI: Gastrin. N Engl J Med 292:1324-1334, 1377-1384, 1975 2. Walsh JH: Radioimmunoassay of gastrin. In Nuclear Medicine in Vitro. Edited by B Rothfield. Philadelphia, JB Lippincott, 1974, p 231-248 3. Walsh JH, Debas HT, Grossman MI: Pure human big gastrin; immunochemical properties, disappearance half time, and acid stimulating action in dogs. J Clin Invest 54477~465,1974