Hepatic Inactivation of Vasoactive Intestinal Peptide in Man and Dog * AMIN M. EBEID, M.D., JEAN ESCOURROU, M.D., PETER B. SOETERS, M.D., PHILIP MURRAY, M.S., JOSEF E. FISCHER, M.D.
In an effort to document the role of the liver in the catabolism of vasoactive intestinal peptide, several different types of experiments were carried out, including: 1) simultaneous measurement of portal and systemic immunoreactive vasoactive intestinal peptide, both in the basal state and following calcium stimulation; 2) by measuring plasma concentrations of immunoreactive vasoactive intestinal peptide before and after portacaval shunt; 3) by measuring plasma VIP before and after portacaval shunt following calcium, prostigmine and pentagastrin stimulation; 4) by determining plasma VIP levels in patients with liver disease and in hepatic failure, and in patients with variceal hemorrhage before and serially after portal systemic shunt; 5) by measuring CSF vasoactive intestinal peptide in dogs before and after portacaval shunt and when the animals finally succumb to hepatic failure. The results consistently suggest that the shunting of portal blood away from the liver does not result in significant elevation of basal peripheral plasma levels of vasoactive intestinal peptide. Following stimulation however, increased amounts of peripheral plasma VIP are detected, following calcium, pentagastrin and prostigmine release of VIP. Portal vein levels are always significantly higher than peripheral plasma VIP again, confirming a catabolic role for the liver. In patients, elevation of peripheral plasma VIP is seen in hepatic failure, but not after portacaval shunt. Finally, cerebrospinal fluid VIP is elevated in dogs following hepatic failure, confirming the presence of a neural-gut axis and suggesting an influence of hepatic catabolism of VIP not only in the periphery, but also within the central nervous system. V ASOACTIVE INTESTINAL PEPTIDE (VIP) is a 28 amino
acid candidate hormone recently isolated by Said and Mutt from the porcine duodenum". It is released into the portal circulation from the entire length of the gastrointestinal tract and possibly from the pancreas as well2'5. Its structure has been determined and its many actions demonstrated' '0'1214 A portion of this paper, "Pentagastrin Release of Vasoactive Intestinal Peptide," was presented at the Tripartite Meeting of the Society of University Surgeons, the British Society for Experimental Surgery and the European Society for Surgical Research, Philadelphia, Pennsylvania, September 27-29, 1976. Reprint requests: Josef E. Fischer, M.D., Massachusetts General Hospital, Boston, Massachusetts 02114. Submitted for publication: July 8, 1977.
Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
Previous work has suggested that the site of VIP inactivation is the liver5"3. The purpose of this study was to determine whether the liver had a role in the inactivation of VIP in the following manner. First: By the simultaneous measurement of plasma VIP levels and portal and systemic vein in the anesthetized dog. If portal vein levels were significantly higher than peripheral levels, hepatic inactivation has occurred. Second: By the study of the effect of portal systemic shunting on basal and stimulated immunoreactive plasma VIP levels in the dog. Third: By determining plasma VIP levels in patients with liver disease and hepatic encephalopathy (presumably on the basis of hepatic failure) and in patients with variceal hemmorhage both before and serially after portal systemic shunt. In this way, at least in man, the contributions of diminished hepatic function and portal systemic shunting to elevation of VIP levels would be determined. Fourth: VIP has been reported as being a normal component in brain3. VIP was measured in the CSF in dogs under normal circumstances and in hepatic encephalopathy, each animal serving as its own control. The effect of hepatic function on brain VIP, if any, would thus be ascertained.
*
Animal Experiments
Methods Mongrel conditioned dogs weighing between 18-22 kg were used throughout. All blood samples were taken with the animal in the fasting state, but not anesthetized
0003-4932-78-0700-0028-0075 © J. B. Lippincott Company
28
Vol. 188oNo. I
VASOACTIVE INTESTINAL PEPTIDE TABLE 1. Plasma VIP In Portal vs. Systemic Vein (5 Normal Dogs) (pglml)
unless otherwise noted. Blood was collected in chilled heparinized tubes, and centrifuged for 15 minutes (3000 rpm) at 40, and the separated plasma frozen at -70° until radioimmunoassay. Basal samples were taken at least twice in each animal.
Simultaneous Determination of Plasma Samples in Portal and Systemic Vein Five dogs were anesthetized with Surital and through a midline laparotomy, the portal vein was cannulated through a small intestinal radical with a catheter threaded proximal into the main portal vein. For the "peripheral venous sample" the left femoral vein was cannulated and the cannula threaded into the inferior vena cava and blood drawn at zero, three, five, ten and 30 minutes following calcium stimulation. Experiments in Animals with End-to-Side Portacaval Shunt Six mongrel dogs (weighing 18-22 kg) were submitted to midline laparotomy under Suritol anesthesia. An end-to-side portacaval shunt was then performed. Clamping time was always less than 20 minutes and the abdomen was closed in two layers. Animals were maintained on intravenous fluids for the first 24 hours and a normal kennel diet thereafter until they went into encephalopathy. Antibiotics (penicillin and streptomycin) were administered for the first 72 hours.
Release Experiments in Shunted Dogs Substances known to release vasoactive intestinal peptide were administered to each animal, before and approximately five weeks after shunt surgery, each animal serving as its own control. These releasing substances were: 1) Calcium 0.25 mEq/kg body weight given in an intravenous drip over a period of 5 minutes, for a total dose of 180 mg of Ca+ + in an 18 kg dog. 2) Neostigmine 0.07 mg/kg given over one minute in 10 ml normal saline. 3) Pentagastrin 0.6,ug/kg given over one minute intravenously in 10 ml normal saline. CSF Levels in Dogs in the Normal State After Shunt and During Encephalopathy Cisternal CSF is thought to be representative of at least some of the metabolism of the canine brain. CSF samples were drawn by cisternal puncture, under light anesthesia (any movement during puncture would be fatal) in six different mongrel dogs of 18-22 kg, before, one and two weeks after end-to-side portacaval shunt and when the animal went into hepatic failure and encephalopathy, generally five weeks after shunt. Statis-
29
Following Calcium Stimulation Basal Values
3'
5'
10'
30'
Portal Vein
240.0 ± 31.6
469.3 ± 60.2
447.0 ± 76.2
503.5 + 105.5
325.3 ± 38.6
Systemic Vein
137.6 + 26.7
196.4 ± 22.8
185.1 ± 27.7
234.7 ± 96.7
150.8 ± 42.6
p
In an effort to document the role of the liver in the catabolism of vasoactive intestinal peptide, several different types of experiments were carried out, including: 1) simultaneous measurement of portal and systemic immunoreactive vasoactive intestinal peptide, both in the basal state and following calcium stimulation; 2) by measuring plasma concentrations of immunoreactive vasoactive intestinal peptide before and after portacaval shunt; 3) by measuring plasma VIP before and after portacaval shunt following calcium, prostigmine and pentagastrin stimulation; 4) by determining plasma VIP levels in patients with liver disease and in hepatic failure, and in patients with variceal hemorrhage before and serially after portal systemic shunt; 5) by measuring CSF vasoactive intestinal peptide in dogs before and after portacaval shunt and when the animals finally succumb to hepatic failure. The results consistently suggest that the shunting of portal blood away from the liver does not result in significant elevation of basal peripheral plasma levels of vasoactive intestinal peptide. Following stimulation however, increased amounts of peripheral plasma VIP are detected, following calcium, pentagastrin and prostigmine release of VIP. Portal vein levels are always significantly higher than peripheral plasma VIP again, confirming a catabolic role for the liver. In patients, elevation of peripheral plasma VIP is seen in hepatic failure, but not after portacaval shunt. Finally, cerebrospinal fluid VIP is elevated in dogs following hepatic failure, confirming the presence of a neural-gut axis and suggesting an influence of hepatic catabolism of VIP not only in the periphery, but also within the central nervous system. V ASOACTIVE INTESTINAL PEPTIDE (VIP) is a 28 amino
acid candidate hormone recently isolated by Said and Mutt from the porcine duodenum". It is released into the portal circulation from the entire length of the gastrointestinal tract and possibly from the pancreas as well2'5. Its structure has been determined and its many actions demonstrated' '0'1214 A portion of this paper, "Pentagastrin Release of Vasoactive Intestinal Peptide," was presented at the Tripartite Meeting of the Society of University Surgeons, the British Society for Experimental Surgery and the European Society for Surgical Research, Philadelphia, Pennsylvania, September 27-29, 1976. Reprint requests: Josef E. Fischer, M.D., Massachusetts General Hospital, Boston, Massachusetts 02114. Submitted for publication: July 8, 1977.
Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
Previous work has suggested that the site of VIP inactivation is the liver5"3. The purpose of this study was to determine whether the liver had a role in the inactivation of VIP in the following manner. First: By the simultaneous measurement of plasma VIP levels and portal and systemic vein in the anesthetized dog. If portal vein levels were significantly higher than peripheral levels, hepatic inactivation has occurred. Second: By the study of the effect of portal systemic shunting on basal and stimulated immunoreactive plasma VIP levels in the dog. Third: By determining plasma VIP levels in patients with liver disease and hepatic encephalopathy (presumably on the basis of hepatic failure) and in patients with variceal hemmorhage both before and serially after portal systemic shunt. In this way, at least in man, the contributions of diminished hepatic function and portal systemic shunting to elevation of VIP levels would be determined. Fourth: VIP has been reported as being a normal component in brain3. VIP was measured in the CSF in dogs under normal circumstances and in hepatic encephalopathy, each animal serving as its own control. The effect of hepatic function on brain VIP, if any, would thus be ascertained.
*
Animal Experiments
Methods Mongrel conditioned dogs weighing between 18-22 kg were used throughout. All blood samples were taken with the animal in the fasting state, but not anesthetized
0003-4932-78-0700-0028-0075 © J. B. Lippincott Company
28
Vol. 188oNo. I
VASOACTIVE INTESTINAL PEPTIDE TABLE 1. Plasma VIP In Portal vs. Systemic Vein (5 Normal Dogs) (pglml)
unless otherwise noted. Blood was collected in chilled heparinized tubes, and centrifuged for 15 minutes (3000 rpm) at 40, and the separated plasma frozen at -70° until radioimmunoassay. Basal samples were taken at least twice in each animal.
Simultaneous Determination of Plasma Samples in Portal and Systemic Vein Five dogs were anesthetized with Surital and through a midline laparotomy, the portal vein was cannulated through a small intestinal radical with a catheter threaded proximal into the main portal vein. For the "peripheral venous sample" the left femoral vein was cannulated and the cannula threaded into the inferior vena cava and blood drawn at zero, three, five, ten and 30 minutes following calcium stimulation. Experiments in Animals with End-to-Side Portacaval Shunt Six mongrel dogs (weighing 18-22 kg) were submitted to midline laparotomy under Suritol anesthesia. An end-to-side portacaval shunt was then performed. Clamping time was always less than 20 minutes and the abdomen was closed in two layers. Animals were maintained on intravenous fluids for the first 24 hours and a normal kennel diet thereafter until they went into encephalopathy. Antibiotics (penicillin and streptomycin) were administered for the first 72 hours.
Release Experiments in Shunted Dogs Substances known to release vasoactive intestinal peptide were administered to each animal, before and approximately five weeks after shunt surgery, each animal serving as its own control. These releasing substances were: 1) Calcium 0.25 mEq/kg body weight given in an intravenous drip over a period of 5 minutes, for a total dose of 180 mg of Ca+ + in an 18 kg dog. 2) Neostigmine 0.07 mg/kg given over one minute in 10 ml normal saline. 3) Pentagastrin 0.6,ug/kg given over one minute intravenously in 10 ml normal saline. CSF Levels in Dogs in the Normal State After Shunt and During Encephalopathy Cisternal CSF is thought to be representative of at least some of the metabolism of the canine brain. CSF samples were drawn by cisternal puncture, under light anesthesia (any movement during puncture would be fatal) in six different mongrel dogs of 18-22 kg, before, one and two weeks after end-to-side portacaval shunt and when the animal went into hepatic failure and encephalopathy, generally five weeks after shunt. Statis-
29
Following Calcium Stimulation Basal Values
3'
5'
10'
30'
Portal Vein
240.0 ± 31.6
469.3 ± 60.2
447.0 ± 76.2
503.5 + 105.5
325.3 ± 38.6
Systemic Vein
137.6 + 26.7
196.4 ± 22.8
185.1 ± 27.7
234.7 ± 96.7
150.8 ± 42.6
p
