Immunochemical Measurements of Endogenous Gastrin Release

GASTROENTEROLOGY Official Publication of the American Gastroenterological Association ©CoPYRIGHT 1970 THE WILLIAMs

VoLUME

59

&

WILKINS Co.

October 1970

NuMBER

4

IMMUNOCHEMICAL MEASUREMENTS OF ENDOGENOUS GASTRIN RELEASE JAMES E. McGuiGAN, M.D. , BERNARD M . JAFFE, M.D., AND WILLIAM T. NEWTON, M.D.

Department of Medicine, University of Florida College of Medicine, Gainesville, Florida, and the Departments of Medicine and Surgery Washington University School of M edicine and John Cochrans Veterans Administration Hospital, St. Louis, Missouri

In anesthetized dogs polyethylene catheters were inserted in the portal vein, hepatic vein, left femoral vein, and thoracic duct after which the pyloric region of the stomach was irrigated for 10 min with 1% (w/v) acetylcholine solution. Gastrin concentrations were measured by radioimmunoassay in venous sera and thoracic duct lymph prior to, during, and following acetylcholine irrigation. Gastrin concentrations were found to increase promptly in the portal vein sera, i.e., within 2 V2 min of initiation of acetylcholine irrigation. Prompt increases in serum gastrin concentrations were also measured in hepatic and peripheral venous blood. Highest concentrations of gastrin were achieved in portal venous sera; the mean gastrin concentrations in hepatic vein sera were 60% of those in portal sera obtained simultaneously. Increases in peripheral venous gastrin concentrations were observed within 10 min of initiation of acetylcholine irrigation. Thoracic duct lymph gastrin levels were consistently less than peripheral venous serum gastrin levels in response to acetylcholine stimulation. These studies do not support the view that the thoracic duct lymph is a major route for conduction of immunoreactive gastrin to the peripheral circulation following acetylcholine stimulation of the stomach. The production of antibodies to the peptide hormone gastrin 1 • 2 and the development of radioimmunoassay techniques3 ' 4 have made it possible to measure gastrin

in physiological fluids and tissues of man and experimental animals. Acetylcholine perfusion of the antrum or antral pouches in experimental animals has been demon-

Received January 19, 1970. Accepted March 26, 1970. Address requests for reprints to: Dr. James E. McGuigan, Department of Medicine, University of Florida College of Medicine, Gainesville, Florida 32601. This work was supported in part by Research

Grant R01 AM 10837 and Training Grant PM 371 from the National Institutes of Health, United States Public Health Service; by Grants T-3948 from The American Cancer Society; and by Veterans Admin· istration research funds. Dr. McGuigan was also supported by Research Career Development Award A1-19,499 from the National Institutes of Health. 499

500

MCGUIGAN ET AL.

strated to be a potent mechanism for release of gastrin activity. s-s The present studies were designed to measure serum gastrin concentrations in portal, hepatic, and peripheral venous sera before and following acetylcholine-provoked release of gastrin from the antral mucosa . In addition, the role of thoracic duct lymph as a possible route of access for gastrin to the peripheral circulation was investigated. Materials and Methods Experimental procedure. Four adult mongrel dogs, from 16 to 22 kg in weight, were utilized for these experiments. The dogs were anesthesized with intravenous Nembutal. Oralgastric tubes were placed so that their emitting orifices were in the gastric antra 5 em above the pylorus. No pylorus ligation was performed. Following surgical entry into the abdominal cavity a polyethylene catheter was placed in the portal vein (via a small tributary) and advanced to the porta hepatis. A hooked-tip cardiac catheter was threaded through a femoral venotomy in the vena cava and positioned in a major hepatic vein. The thoracic duct was identified for purposes of catheterization by injections of Evans blue dye into mesenteric lymph nodes with detection of the subsequent appearance of the readily visible blue dye in the cervical region of the thoracic duct. The thoracic duct was ligated and a polyethylene cannula threaded caudad for 3 to 5 em . The left femoral vein was also cannulated. After the abdomen was closed the stomach of each dog was irrigated with physiological saline (0.15 M NaCl) following which the preparation was allowed to equilibrate for 2 hr. An acetylcholine chloride solution (1 % w/v) was then gently irrigated into the antral portion of the stomach at a rate of 50 ml per min for a 10min period. At each min 50 ml were introduced irrigated, and aspirated. For the final 3 mi~ of the 10-min period approximately 75 ml of acetylcholine solution were allowed to remain in the stomach. Irrigation with acetylcholine was then discontinued and the oral-gastric tube was allowed to drain freely. More than 90% of the acetylcholine solution was retrieved during and immediately following irrigation. After 5 ml of fluid had been aspirated from each catheter and were discarded, blood samples (5 ml) were collected from each venous catheter immediately prior to the initiation of acetylcholine irrigation, which was designated time zero, and at 2.5-, 5, or 10-min intervals thereafter for a total time duratiOn of 50 min. Tho-

Vol. 59, No.4

racic duct lymph was collected in separate aliquots for 10 min prior to initiation of acetyl. choline perfusion, for the 10-min interval dur. ing which acetylcholine was perfused, and for four subsequent 10-min intervals. At the con. elusion of the experiments proper positioning of the cannulas was verified. Radioimmunoassay of gastrin. Concentrations of gastrin in serum and in thoracic duct lymph were measured as previously described utiliz. ing a "double antibody" radioimmunoassay technique. 3 ' ·• Antibodies utilized for radioimmunochemical measurement of gastrin were those in the serum obtained from 1 rabbit following repeated immunization with synthetic human gastrin I (residues 2 through 17) covalently conjugated to bovine serum albumin. The antibody-containing serum was obtained from the rabbit by cardiac puncture 16 months following initial immunization. This selected antibody preparation evoked by immunization with human gastrin I exhibited almost identical binding of canine gastrin I when compared with its binding of human gastrin I (fig. 1) . (Pure canine gastrin I was kindly supplied by Professor R. A. Gregory, University of Liverpool.) Through the range of measurement the ratio of immunoreactivity of human to canine gastrin I was from 1.1 to 1 to 1.3 to 1. Therefore, calibration diagrams were established utilizing human gastrin I and expressed as immunoreactive gastrin equivalent to human gastrin I. Calibration diagrams using dilutions of gastrin in standard ovalbumin- and serum-containing solutions' were used for serum measurements.

50

'0

\_

l!! 40

.E :§. u ~

Q

.?:-

30

~ 0

j *

.,

\

20

10

---1

10

100

1000

Picogroms Gastrin

FIG. 1. Radioimmunoassay calibration diagrams comparing immunoreactivity of human gastrin I (e) and canine gastrin I (Q) using antibodies to human gastrin I (residues 2 through 17) and "'I· human gastrin I.

October 1970

ENDOGENOUS GASTRIN RELEASE

In addition, calibration diagrams for measurement of gastrin in thoracic duct lymph were established using synthetic human gastrin I diluted in fasting thoracic duct lymph from 1 dog in which gastrin was immeasureably low (this dog was separate from those described in these studies). Serum and thoracic duct lymph gastrin concentrations were measured in triplicate by radioimmunoassay on two or three separate occasions; the results expressed in these studies represent the means of those determinations. In the determination of precision of analysis the mean standard errors for triplicate determinations were 17 and 12% for 5 to 50 and 50 to 1000 pg, respectively.

Results Peripheral venous gastrin concentrations. Mean peripheral venous gastrin concentrations for the 4 dogs are displayed in figure 2. The mean prestimulated fasting serum gastrin concentration for the 4 dogs was 80 pg per ml. Peak peripheral venous gastrin concentrations, for individual dogs following stimulation with acetylcholine, varied from 293 to 792 pg per ml. For each of the 4 dogs increases in peripheral venous serum gastrin were detected at the earliest point in observation which was 10 min following the initiation of acetylcholine infusion at which time irrigation was terminated. Higher mean levels at 40 and 50 min (fig. 2) reflect in principal part a late appearing sharply elevated second peak in 1 of the 4 dogs (dog 4). Thoracic duct lymph concentrations. The mean prestimulation thoracic duct lymph concentration was 88 pg per mi. Thoracic duct lymph concentrations were expressed as mid points in the collection time periods (fig. 3). Following acetylcholine stimulation for all measurements in all 4 dogs thoracic duct lymph gastrin concentrations were less than those found at corresponding times in peripheral venous serum. The mean peak thoracic duct lymph concentration was 232 pg per ml which was achieved in lymph collected during the interval from 20 to 30 min following initiation of acetylcholine infusion of the antral portion of the stomach.

501

800·r-----------------------------, :::::-

Acetylcholine

Infusion

~ 600

"'

i:)~P:~I~ J

0~--~----~----~----L---~~

0

10

20

30

«l

50

MINUTES

FIG. 2. Mean peripheral venous serum gastrin concentra tions before and following lO·min infusion of acetylcholine in all 4 dogs. (Bracket ed vertical bars represent ± 1 standard error of the mean .)

In each of the 4 dogs the thoracic duct lymph levels appeared to reflect peripheral venous gastrin levels but were somewhat less in concentration and delayed in time when compared with those of peripheral sera. Portal venous gastric concentrations. The mean portal vein serum gastrin concentrations from the 4 dogs are demonstrated in figure 4. The mean portal venous serum gastrin level prior to stimulation with acetylcholine was 212 pg per ml, higher than that for the mean gastrin concentration in prestimulation peripheral sera (80 pg per ml). The mean portal vein serum gastrin increased from 212 pg per ml, at which time acetylcholine irrigation was initiated, to 805 pg per ml following 10 min of infusion. Mean portal serum gastrin concentrations subsequently increased to peak levels at 35 and 50 min. Hepatic venous serum gastrin concentrations. Mean hepatic venous serum gastrin concentrations are displayed and compared with portal venous serum gastrin concentrations in figure 4. The mean prestimulation hepatic venous serum gastrin concentration was 125 pg per ml, higher than that of peripheral venous serum but less than that of the portal vein serum. Hepatic vein serum gastrin levels increased promptly following initiation of gastric acetylcholine irrigation

502

Vol. 59, No.4

MCG UIGAN ET AL.

eoo,---------------------------------------------------~

W////#o??:?:i'l ACETYLCHOLINE

600

INFU SION

thoraCIC duct lymph

5

~--1-------J- ~ --·2/ 0 ~~--~o~--~--~ ~o--~--~2~o--~--~3~ o ---L---4~o----~--Jso~

MINUTES FIG . 3. Mean thoracic duct lymph gastrin concentrations before and following 10-min infusion of acetylcholine in all 4 dogs. (Brack et ed vertical bars represent ± 1 standard error of the mean.)

1400 ~1200 ~1000 ~

<:

800

~

600

~

::;: 400

~

~200 0

0

10

20

30

40

50

MINUTES

FIG . 4. Mean portal venous and hepatic venous gastrin concentrations before and followin g 10-min infusion of acetylcholine in all 4 dogs. (Brack eted vertical bars represent ± 1 SE.)

with the highest mean hepatic vein serum level (540 pg per ml) being achieved 30 min following initiation of stimulation with acetylcholine. Portal and hepatic serum gastrin concentrations for all observation time points for each of the 4 dogs were compared and expressed as the ratio of portal vein serum gastrin concentration to hepatic vein serum gastrin concentration. This ratio was found to be 1. 7 ± 0.32 (SE)

Discussion These studies, for the first time, directly demonstrate circulating gastrin in both portal and peripheral blood following stimulation of the pyloric antrum . Although the stomach was aspirated and drained following irrigation there was no attempt to exclude completely the acetylcholine solution from the duodenum, as, for instance, by pyloric ligation. Therefore, the possibility remains that a portion of the circulating gastrin measured in these studies may have been released from sites in addition to the antrum, e.g., from the duodenum. Acetylcholine perfusion of the gastric antra of dogs has been demonstrated to be an effective method for stimulation of the release of gastrin activity as evidenced by resultant stimulation of gastric acid secretion. 5 _8 Cooke et al. 7 have found that the maximal acid output of canine gastric fistulas evoked by perfusion of antral pouches approximated those for maximal acid output following intravenous administration of hog gastrin or histamine. Cooke and Grossman 8 have recently described acid secretory responses in dogs with transplanted and extrinsically de-

October 1970

ENDOGENOUS GASTRIN RELEASE

nervated antral pouches, gastric fistulas, and Heidenhain pouches. Gastric acid secretory responses to antral distension and perfusion with acetylcholine chloride, ethanol, meat extracts, glycine, sodium chloride, and sodium bicarbonate solutions were measured and compared with maximal responses to intravenous histamine. Acid output from canine gastric fistuals and Heidenhain pouches was shown to increase within 15 min following irrigation of antral pouches with acetylcholine. Antral pouch acetylcholine perfusion evoked rates of gastric acid secretion from gastric fistuals which were 3 times those achieved with perfusion with a 7.5% glycine solution. In that study peak acid output from gastric fistuals was almost as great (95%) with acetylcholine perfusion of the antrum as maximal responses following intravenous histamine. Among the substances used to perfuse the canine antral pouches acetylcholine was found to be the most potent stimulant of acid secretion, both from gastric fistulas and Heidenhain pouches. The studies described in this report demonstrate directly, by radioimmunoassay, increases in concentrations of gastrin in portal, hepatic, and peripheral venous serum following instillation of 1% acetylcholine solution into the stomach with a catheter tip placed in the gastric antrum. Increases in portal serum gastrin concentrations were detected promptly, being identified at the earliest time points of observation during stimulation, i.e., at 2 1/ 2 min following initiation of acetylcholine irrigation. Increases in gastrin concentration in peripheral venous serum were recognized at the earliest point of observation which was 10 min following the initiation of acetylcholine irrigation. Peripheral serum gastrin concentrations, both before and following irrigation with acetylcholine, were found to be substantially less than those of portal vein serum. Hepatic vein serum gastrin levels also increased promptly during and following irrigation of acetylcholine into the antral portions of the stomachs. Hepatic venous serum gastrin levels exceeded those of peripheral ven-

503

ous sera and were approximately 60% of those in portal venous sera. These studies confirm prior physiological studies demonstrating that gastrin is released from the stomach following local acetylcholine infusion and indicate it is transported via the portal vein to the liver and thence to the peripheral circulation through hepatic venous channels. These studies also demonstrated the presence of, and increases in, concentration of gastrin in thoracic duct lymph following acetylcholine infusion. The finding of gastrin in lymph is not unique inasmuch as other hormones have been found in lymph: thyroid hormone 9 has been found in lymph draining the thyroid gland and renin and an angiotensin-like material 10 have been found in renal lymph. Gastrin concentrations in thoracic duct lymph before, during, and following acetylcholine infusion were substantially less than those found in portal vein sera. Prior to acetylcholine stimulation thoracic duct lymph gastrin concentrations were comparable to those found in peripheral venous sera. Concentrations of thoracic duct lymph gastrin, which increased following acetylcholine infusion, were less than, and perhaps delayed in time, when compared with peripheral venous serum gastrin concentrations. Studies of the distribution of 1t 5 I human gastrin following intravenous administration into dogs indicate its distribution into a space which approximates 19% of body weight, a volume comparable to the extracellular compartment. 11 It is reasonable, therefore, to conclude that increases in thoracic duct gastrin levels observed in these studies following acetylcholine perfusion probably reflect, at least in part, extracellular gastrin. Rudick et al. 1t detected the presence of a gastric acid secretagogue in the thoracic duct lymph of dogs acetylcholine perfusion of denervated antral pouches. Others 1a have found a gastric acid secretagogue in canine thoracic duct lymph collected following feeding: this acid secretagogue was not present in the thoracic duct lymph of antrectomized dogs following feeding. The great disparity between rates of thoracic duct lymph flow

504

MCGUIGAN ET AL.

Vol. 59, No. 4

and portal venous flow and the substan2. Gregory RA: Memoria l Lecture. The isolation and chemistry of gastrin. Gastroenterology 51: tially greater gastrin concentrations in 953-959, 1966 portal vein blood compared with those 3. McGuigan JE: Immunochemical studies with in thoracic duct lymph indicate that any synthetic human gastrin. Gastroenterology 54: contribution to the stimulation of gastric 1005-1011, 1968 acid secretion must be small. During the 4. McGuigan JE, Trudeau WL: Studies with antiperiod of measurement of gastrin levels bodies to gastrin: radioimmunoassay in human during and following acetylcholine stimuserum and physiological studies. Gastroenterollation in the studies described in this ogy 58:139-150, 1970 report the quantity of gastrin transported 5. Robertson CR, Langlois K, Martin CG, et a!: Release of gastrin in response to bathing the via the thoracic duct was less than 1% pyloric mucosa with acetylcholine. Amer J of that transported via the portal vein. Physiol163:27-33, 1950 Thus, although showing an increase in 6. Olbe L, Elwin C: Mechanisms Stimulating Angastrin in thoracic duct lymph following tral Release of Gastrin. Postgraduate Gastrostimulation with acetylcholine, the reenterology. Edited by TJ Thompson, IE Gilsults of these studies do not support lespie. London, Balliere, Tindall and Cassell, thoracic duct lymph as a major route for 1966 transportation of gastrin from the stomach 7. Cooke AR, Nahrwold DL, Preshaw RM, et a!: to the peripheral circulation. It is likely Comparison of endogenous and exogenous gasthat gastrin in thoracic duct lymph reptrin in stimulation of acid and pepsin secretion. resents equilibration of gastrin molecules Amer J Physiol 213 :432-436, 1967 8. Cooke AR, Grossman MI: Comparison of stimuwithin the extracellular fluid compartlants of antral release of gastrin . Amer J Physiol ment. 215:314-317, 1968 The lower levels of hepatic serum gas9. Daniel PM, Excell BJ, Gale MM, et a!: The trin when compared with those of portal drainage of thyroid hormone by the lymphatics venous serum reflect, in great part, diluof the thyroid gland. J Physiol (London) 160:6, tion of portal vein blood with hepatic ar1962 terial blood. 14 - 16 These studies do not sup- 10. Lever AF, Peart WS: Renin and antiotensin-like port the view that substantial extraction activity in renal lymph. J Physiol (London) of gastrin occurs during its transport 160:548-553, 1962 through the liver. It must be pointed out 11. Jaffe BM, Newton WT: Distribution and localization of radioiodinated gastrin. Surg Forum that these studies describe the measure20:312-313, 1969 ment of immunoreactive gastrin which may or may not correspond precisely 12. Rudick J , Fletcher TL, Dreiling DA: Effects of lymph diversion on the gastric secretory response with physiologically active gastrin moleto endogenous gastrin. Amer J. Physiol 215: cules. Gastrin activity may be substan370-373, 1968 tially reduced or abolished by relatively 13. Kelly KA, Ikard RW, Nyhus LM , eta!: Gastric slight chemical modification of the carsecretagogues in postprandial thoracic duct boxyl portion of the hormone which conlymph. Amer J Physiol 205:85-88, 1963 tains the physiologically active site of the 14. Burton-Opitz R: The vascularity of the liver. hormone; e.g., deamidation ofthe carboxylII. The influence of the portal blood-flow upon terminal phenylalanine amide results in the flow in the hepatic artery. Quart J Exp almost complete abolition of gastrin acPhysiol 4:93-102, 1911 tivity . 17 At present it is not known to 15. Bauer W, Dale HH, Poulsson LT, et a!: The what extent deamidation of the gastrin control of circulation through the liver. J Physiol (London) 74:343-375, 1932 molecule affects its immunoreactivity. REFERENCES 1. Gregory RA, Tracy HF: The constitution and properties of two gastrins extracted from hog antral mucosa. I. The isolation of two gastrins from hog antral mucosa. II. The properties of two gastrins isolated from hog mucosa. Gut 5:103-114, 1964

16. Schwenk WG Jr, McDonald JC, McDonald K, et a!: Direct measurement of hepatic blood flow in surgical patients: with related observations on hepatic flow dynamics in experimental animals. Ann Surg 156:463-471, 1962 17. Morley JS: Structure-function relationships in gastrin-like peptides. Proc Roy Soc London SerB 170:97-111, 1968