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Postprandial sphincter of Oddi myoelectric activity and gallbladder emptying
JOURNAL
OF SURGICAL
RESEARCH
Postprandial
4,
596-602 (1988)
Sphincter of Oddi Myoelectric and Gallbladder Emptying’
Activity
JOSE E. PARODI, M.D., JAMES M. BECKER, M.D., DAVID I. SOYBEL, M.D., BART BARRUS, B.S., AND PAUL E. CHRISTIAN, B.S. Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110; and Department of Nuclear Medicine, University of Utah School of Medicine, Salt Lake City, Utah Presented at the Annual Meeting of the Association for Academic Surgery, Orlando, Florida, November 1-4, 1987 Feeding initiates gallbladder emptying and bile delivery into the duodenum. It is not yet defined how the sphincter of Oddi regulates flow of bile into the duodenum during gallbladder emptying. The aim of this study was to assesspostprandial spike burst activity in the sphincter of Oddi, while quantitating gallbladder emptying with noninvasive radioisotope imaging. Six adult opossums were prepared with bipolar electrodes in the sphincter of Oddi. After 2 weeks of recovery the animals were fasted overnight and positioned under a gamma camera, and myoelectric recordings were begun. After two cycles of the migrating motor complex (MMC), 2 mCi 99Tc-HIDA was infused intravenously and permitted to concentrate in the gallbladder for a period of 30 min. Subsequently, a 30-ml liquid meal, containing 0.9 g protein, 3.5 g carbohydrate, and 3.3 g fat, was instilled into the stomach. Sphincter of Oddi myoelectric activity (spike bursts/min) and gallbladder emptying (expressed as percentage of original 99Tc counts in the gallbladder) were measured at intervals for 120 min following feeding. Feeding resulted in prompt gallbladder emptying. Sphincter of Gddi spike burst activity was not altered significantly in the first 30 min after feeding, suggesting that motor activity in the sphincter of Oddi does not initially influence bile flow. Subsequently, spike burst activity increased progressively, suggestingthat sphincter of Oddi motor activity may accelerate bile delivery into the duodenum during later phasesofgallbladder emptying. 0 1988 Academic Press, Inc.
INTRODUCTION
The use of radionuclide imaging to study biliary motility in vivo has been reported in animals and humans [ l-51. Using these techniques it is possible to study the interaction of gallbladder emptying with motor activity of the biliary sphincter of Oddi. It has been postulated [6, 71 that such interactions may regulate the flow of bile into the duodenum following feeding. In this study we used radionuclide imaging techniques to quantitate postprandial gallbladder emptying while monitoring electrical spike burst activity of the sphincter of Oddi in the opossum. The results suggestthat motor activity in the biliary sphincter may not only restrict flow of bile into the duodenum before feeding, but also may accelerate emptying of bile from ’ Supported by NIH Grant 2-RO1-DK3583 l-05. 0022-4804/88 $1.50 Copyri8ht Q 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
the gallbladder into the duodenum during the late postprandial period. METHODS
Studies were carried out in 10 adult opossums (Didelphis virginiana, R-Zoo, Neshkoro, WI) of either sex and parasite free. The animals were anesthetized with sodium pentobarbital (20-30 mg/kg) injected intraperitoneally and maintained with small intravenous pulses as needed. Using sterile surgical technique a midline abdominal incision was made and the biliary tree was exposed, The sphincter of Oddi was identified as a thick smooth muscle structure l-2 cm in length extending from the junction of the common bile duct and pancreatic duct (proximal sphincter) to the duodenal wall (distal sphincter). Bipolar insulated copper wire (0.12 in. in o.d., Biomed Wire, Cooner Co.,
596
PARODI ET AL.: SO AND GALLBLADDER
Chatworth, CA) electrodes were secured to the smooth muscle of the proximal and distal sphincter (Fig. 1). A monopolar shield electrode was placed within the duodenal wall 10 cm distal to the choledochoduodenal junction to serve as a ground. The electrode wires were brought out through the abdominal incision, tunneled subcutaneously to the midscapular region, and brought out through a transcutaneous stainless steel cannula fitted with an Amphenol-five pin connector. The electrodes themselves were constructed by removing a 2-mm band of insulation, 4 cm from the end of the electrode wire. The electrode was introduced into the tissue by threading the wire through a 23-gauge hypodermic needle passed through the circular layer of muscle. The bare section of the wire was positioned within the tissue and the free exiting end was tied to the fixed entering end. The abdominal wall and skin were then closed and a longitudinal incision was made in the right anterolateral neck. The right jugular vein was dissected and a 9-Fr Teflon catheter with a vascular port (Model GPI, Norfolk Medical, Inc., Stokie, IL) was inserted in the vein and was placed in a subcutaneous pocket. After a 2-week recovery period, each animal was fasted overnight and a slow intravenous saline infusion was started (0.5 ml/min)
FIG. 1. Chronic experimental preparation in the opossum. Bipolar wire electrodes are secured to the proximal and distal sphincter of Oddi.
EMPTYING
597
through the vascular port. The animals then received pancuronium bromide (0.1 mg/kg) intravenously, and were intubated endotracheally and ventilated mechanically. Paralysis was maintained with intravenous pulses of pancuronium bromide. Skeletal muscle paralysis was utilized to eliminate motion artifact on the radioimaging recordings. The electrocardiogram of the animal was monitored throughout each experiment. Arterial blood gas analysis was performed intermittently to assure the consistency of mechanical ventilation. The output of the biliary electrode was connected to an eight-channel Gould chart recorder (Gould Electronics Division, Cleveland, OH). The polygraph was calibrated to record slow waves with a low cutoff filter of 0.16 Hz, and spike bursts with a high cutoff filter of 30- 100 Hz. Myoelectric activity of the sphincter of Oddi was recorded continuously using a chart speed of 1 mm/set. After recording two cycles of the interdigestive motor complex (MMC), the abdomen was centered over the gamma camera and 2 mCi of 9gTc-HIDA was infused via the vascular port. The isotope was permitted to concentrate in the gallbladder for 30 min before each experimental protocol was begun. Gamma radiation over the abdomen was measured with a medium energy collimator. Serial analog images were processed every minute by a Techni-Care T60 computer system and stored on a floppy disc. When the gallbladder was well visualized with radioisotope (30 min, in all cases)a 6-mm orogastric tube was placed and a liquid test meal composed of 3.3 g of fat, 3.5 g of carbohydrate and 0.9 g of protein, with a total volume of 30 ml (long chain triglycerides, Sandoz Pharmaceuticals Corp., NJ, and Enrich Source Fiber, Ross Laboratories, Division of Abott Laboratories, Columbus OH), was instilled into the stomach. Radionuclide imaging was continued for 4-6 hr until at least 50% of the radioisotope had disappeared from the gallbladder and appeared in the duodenum. The pancuronium bromide was reversed with neostigmine (0.25 mg intrave-
598
JOURNAL OF SURGICAL RESEARCH: VOL. 44, NO. 5, MAY 1988
nously and repeated as needed) and subcutaneous atropine and the animal was allowed to recover.
0.6-J
i
o[
1 0
I 30
i 60
I 90
! I20
TIME 1min 1 FIG. 3. Time activity curve of 99Tc-HIDA”within the gallbladder in a typical experiment. tJo represents the time required for 50% depletion of gallbladder counts.
Four of the ten opossums were used to determine the effect of pancuronium bromide on sphincter of Oddi myoelectric activity. After an overnight fast, myoelectric activity was recorded for 2 MMC cycles. Intravenous pancuronium was then administered as described above and recordings were continued for 4 to 6 hr, covering at least two further cycles of the MMC. The muscle relaxant was then fully reversed. Myoelectric recordings were visually inspected and the spike bursts per minute were counted and plotted as a histogram. The mean length of the first two MMC cycles and the mean spike bursts per MMC before and after pancuronium were computed and statistically compared using the Student t test for paired data.
ComputerAnalysis of Radioactivity The data were stored on floppy discs at 1-min intervals and anatomic regions of interest, i.e., liver, gallbladder, and duodenum, were outlined on the screen of the computer terminal using a mouse. Counts in each region were plotted on a time activity curve. FIG. 2. Serial scintigram, from the first hour of a typiFrom the curve the percentage of counts cal experiment. Left column represents actual scintiemptied by the gallbladder was calculated grams; right column delineates area of interest selected for measurement of actual counts. Rapid uptake of iso- from the equation: tope by the liver and concentration in the gallbladder in the fasting state are noted (top panel). Animals were fed at 30 min. After feeding, rapid emptying occurs and radionuclide appears within the duodenum.
GB cpm before meal - GB cpm after meal x IO0 GB cpm before meal *
PARODI ET AL.: SO AND GALLBLADDER
EMPTYING
599
TABLE I POSTPRANDIALSPHINCTER OFODDI MYOELECTTRICACTIVITY AND GALLBLADDEREMPTYING Time (mitt) 0
meal
OGBemptied 9.3 + 2.1 PSOspikefrequency (meanSB/IOmitt) 23.3f 3.1 DSOspikefrequency (meanSB/10min) 19.8f 3.7
10
20
30
40
60
50
9.7 2 3.2 21.9+ 4.3*.** 25.4+ 5.9* 31.1+ 6.F
34.0+ 7.v
38.3?I 8.5*
24.6+ 3.4 31.3k 3.0
32.12 2.7 35.5+ 4.1*v** 35.0k 3.1*.*** 31.2+ 2.7
21.4+ 0.3 25.0+ 4.2
25.5k 0.2
30.7f 5.1**
29.9+ 4.8
24.8f 3.7
Nole.Percentage of gallbladderemptyingandsphincterof Oddi mycelectricactivity afterintragastticinstillationof a meal.Values areexpressedasmeans+ SEM. * P < 0.05comparedwith control. l * P < 0.05compared with preceedingtime period. *** P c. 0.05comparedwith first interval afterfeeding.
Analysis of Myoelectric Recordings
shown in Fig. 2. A time activity analysis for a typical experiment is shown in Fig. 3. As illustrated in the figures the marker was rapidly concentrated within the gallbladder, reaching a maximum at 20-30 min after infusion. When the meal was administered a prompt decrease in gallbladder counts was observed. Maximum gallbladder emptying occurred between 10 and 20 min after the meal; by the end of that interval 2 1.9 + 4.3% of gallbladder counts had emptied (n = 10, P < 0.05 compared to before feeding, Table 1).
Recordings of myoelectric activity in the biliary sphincter were inspected and total spike burst frequency within IO-min intervals was analyzed. The data from the gallbladder radionuclide activity curve and sphincter of Oddi spike burst frequencies were entered in a worksheet data base on a Lotus l-2-3 program (Lotus Development Corp., Cambridge MA) on an IBM PC/AT. Statistical analyses were performed using Student’s t test for paired or unpaired data where appropriate. A P level of co.05 was Sphincter of Oddi Myoelectric Activity accepted as significant.
Comparison of fasting myoelectric activity in the presence and absence of intravenous Gallbladder Emptying pancuronium is shown in Table 2. No statistically significant changes were observed in Serial scintigraphs of the abdomen followspike burst frequency and duration of the ing intravenous infusion of 99Tc-HIDA are RESULTS
TABLE 2 EFFECTOFPANCURONIUMBROMIDEON FASTINGMYOELEC~RICACTIVITY OFTHE OPOSSUMSPHINCTEROFODDI Before PSO MMC cycle length (min) DSO MMC cycle length (min) PSO spike bursts/cycle DSO spike bursts/cycle Note. n = 4. ns = not significant.
98.7 +- 6.6 104.3 + 7.3 288.7 + 26.0 223.3 +_22.8
After 91.7 f 104.0 + 263.3 f 209.2 k
5.1 7.9 20.0 26.5
ns ns ns ns
600
JOURNAL OF SURGICAL RESEARCH: VOL. 44, NO. 5, MAY 1988
MEAL
Y 4-
iIs ::1-1
+
2
3
4
5
6
7
6
TIME (hours) FIG. 4. Spike burst frequency histogram, based on S-min interval averages for the proximal and distal sphincter of Oddi prior to and after intragastric meal instillation.
MMC in the sphincter of Oddi. Administration of the fat-containing meal disrupted the interdigestive myoelectric cycles and resulted in an overall increase in sphincter spike burst activity (Fig. 4). Figure 5 compares the time course of changes in myoelectric activity of the proximal and distal sphincter with the time course of gallbladder emptying as obtained by radionuclide imaging. Proximal and distal sphincter spike burst activity did not change during the first IO-min interval after feeding, and thereafter, sphincter spike frequency increased but did not reach significance throughout the first 30-min postprandial interval (Table 1). During the next 30min interval, spike burst frequencies increased significantly in conjunction with progressive emptying of 99Tc-HIDA from the gallbladder. Gallbladder emptying was sustained between 60 and 150 min after feeding and resulted in a depletion of more than 50% of counts. Thus, maximum myoelectric activity of the biliary sphincter was observed not during gallbladder filling before feeding, or at the initiation of gallbladder contraction, but well after the phase of the most rapid postprandial gallbladder emptying. DISCUSSION
Technetium-99-HIDA imaging has been used as a sensitive method for evaluating gallbladder function and disease due to its
high uptake and biliary excretion in both animals and humans. It has been demonstrated in humans and dogs that emptying of bile from the gallbladder is in concert with the later phase II of the migrating motor complex of the small intestine [S]. Also, a reflex interaction of the gallbladder, duodenum,
3 LL
01
30
60 so TIME (minutes)
120
J
I?io
FIG. 5. Simultaneous g9Tc-HIDA time activity curve within the gallbladder (top panel) and proximal sphincter of Oddi (PSO) and distal sphincter of Oddi (DSO) spike burst frequencies (bottom panel) observed before and after intragastric meal instillation. (Values are expressed as means + SEM of IO-min intervals for experiments, n = 10.)
PARODI ET AL.: SO AND GALLBLADDER
and sphincter of Oddi has been suggestedin the regulation of sphincter function and gallbladder motility [6]. Similarly, a relationship between gallbladder evacuation and motor activity of the sphincter has been suggested. It has been demonstrated that in the opossum cholecystokinin (CCK) increases both gallbladder emptying and sphincter of Oddi myoelectric spike activity in a dose-dependent fashion [9-131. This is similar to the effect of CCK in the sphincter of Oddi of the prairie dog, rabbit, and, in some cases,man, and is different from the response observed in the cat, guinea pig, and dog [ 121.The relationship between myoelectric activity of the sphincter of Oddi and gallbladder emptying has not been systematically studied. Using the methodology described above we quantitated gallbladder emptying after instillation of a test meal into the stomach. Our results indicated that the gallbladder of the opossum emptied promptly immediately after feeding. Sphincter of Oddi myoelectric activity remained unchanged during this early postprandial period and did not significantly accelerate until 40 min after feeding. These findings suggesta delay in sphincter of Oddi activation compared to the onset of gallbladder emptying. The observations may have been influenced by the skeletal muscle relaxant employed to eliminate motion artifact; however, our studies demonstrated no effect of pancuronium on biliary myoelectric activity. Additionally, pancuronium has been previously shown to have minimal influence on gastrointestinal smooth muscle [14, 151. The delay in increased spike burst activity in the biliary sphincter suggests that early after feeding, in response to enteric content, bile flow into the duodenum is largely dependent upon contraction of the gallbladder with the sphincter of Oddi playing a minor role in modulating bile flow. In the late postprandial period, as gallbladder emptying slows and overall bile flow declines, sphincter motility increases. During this period the sphincter may serve to actively pro-
EMPTYING
601
pulse bile into the duodenum. Alternatively, the sphincter may simply be responding secondarily to increased postprandial biliary flow and pressure, serving as a meter or valve. Data supporting both hypotheses in many animal species, including man, exist [9, 10, 16-l 81. Further studies monitoring gallbladder and sphincter of Oddi myoelectric activities, bile flow, and biliary pressure are warranted. REFERENCES 1. Engler-t, E., and Chiu V. Quantitative analysis of human biliary evacuation with radioisotopic technique. Gastroenterology 50: 506, 1966. 2. Harvey, E., Loberg, M., Ryan, J., et al. Hepatic clearance mechanism of Tc-99-HIDA and its effect on quantitation of hepatobiliary function. J. Nucl. Med. 20: 310,1979. 3. Ivy, A. C., and Bergh, G. A. The applied physiology of the extrahepatic biliary tract. JAMA 103: 2500,
1934. 4. Krishnamurthy, G. T., Bobba, V. R., and Kingston,
E. Radionuclide ejection fraction: A technique for quantitative analysis of motor function of the human gallbladder. Gastroenterology 80: 482, 1981. 5. Wistow, B. W., Subramanian, G., Vanheeertum, R. I., et al. An evaluation of Tc-99m-labeled hepatobiliary agents. J. Nucl. Med. 18: 455, 1977. 6. Muller, E. L., Lewinski, M. A., and Pitt, H. A. The cholecysto-sphincter of Oddi reflex. J. Surg. Rex 36: 377,1984. I. Takahashi, I., Kern, M. K., Dodds, W. J., Hogan, W. J., Sama, S. K., Soergel, K. H., and Itoh, Z. Contraction pattern of opossum gallbladder during fasting and after feeding. Amer. J. Physiol. 250: G227, 1986. 8. Itoh, Z., and Takahashi, I. Periodic contractions of
the canine gallbladder during the interdigestive state. Amer. J. Physiol. 240: G 183, 1981. 9. Becker, J. M., Moody, F. G., and Zinsmeister, A. R. Effects of gastrointestinal hormones on the biliary sphincter of Oddi of the Opossum. Gastroenterology 82:1300,1982.
10. Behar, J., and Biancani, P. Effect of cholecystokinin and the octapeptide of cholecystokinin on the feline sphincter of Oddi and gallbladder. J. Clin. Invest. 66:1231,1980.
11. Behar, J., and Biancani, P. Pharmacologic characterization of excitatory and inhibitory cholecystokinin receptors of the cat gallbladder and sphincter of Oddi. Gastroenterology 92: 764, 1987.
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12. Sarles, J. C. Hormonal control of sphincter of Oddi. Dig. Dis. Sci. 31: 208, 1986. 13. Spellman, S. J., Shaffer, E. A., and Rosenthal, L. Gallbladder emptying in response to cholecystokinin. A cholescintigraphic study. Gastroenterology 7: 115, 1975. 14. Bromage, P. R. Pulmonary complications of regional anesthesiafor obstetrics. In S. M. Shnider and G. Levinson (Eds.), Anesthesia for Obstetrics, 2nd ed. Baltimore: Williams & Wilkins, 1987. 15. Scott, R., and Savarese, J. The cardiovascular and autonomic effects of neuromuscular blocking agents. In R. L. Katz (Ed.), Muscle Relaxants: Basic
and Clinical Aspects. New York: Grune & Stratton, 1985. 16. Ashkin, J. R., Lyon, D. T., Shuli, S. C., Wagner, C. I., and Soloway, R. D. Factors affecting delivery of bile to the duodenum in man. Gastroenterology 74: 560, 1978. 17. Scott, G. W., Smallwood, R. E., and Rowlands, S. Flow through the bile duct after cholecystectomy. Surg. Gynecol. Obstet. 240: 912, 1975. 18. Tansy, M. F., Innes, D. L., Martin, J. S., and Kendall, F. M. A functional description of canine choledochoduodenal flutter valve. Amer. J. Dig. Dis. 21: 223, 1976.
OF SURGICAL
RESEARCH
Postprandial
4,
596-602 (1988)
Sphincter of Oddi Myoelectric and Gallbladder Emptying’
Activity
JOSE E. PARODI, M.D., JAMES M. BECKER, M.D., DAVID I. SOYBEL, M.D., BART BARRUS, B.S., AND PAUL E. CHRISTIAN, B.S. Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110; and Department of Nuclear Medicine, University of Utah School of Medicine, Salt Lake City, Utah Presented at the Annual Meeting of the Association for Academic Surgery, Orlando, Florida, November 1-4, 1987 Feeding initiates gallbladder emptying and bile delivery into the duodenum. It is not yet defined how the sphincter of Oddi regulates flow of bile into the duodenum during gallbladder emptying. The aim of this study was to assesspostprandial spike burst activity in the sphincter of Oddi, while quantitating gallbladder emptying with noninvasive radioisotope imaging. Six adult opossums were prepared with bipolar electrodes in the sphincter of Oddi. After 2 weeks of recovery the animals were fasted overnight and positioned under a gamma camera, and myoelectric recordings were begun. After two cycles of the migrating motor complex (MMC), 2 mCi 99Tc-HIDA was infused intravenously and permitted to concentrate in the gallbladder for a period of 30 min. Subsequently, a 30-ml liquid meal, containing 0.9 g protein, 3.5 g carbohydrate, and 3.3 g fat, was instilled into the stomach. Sphincter of Oddi myoelectric activity (spike bursts/min) and gallbladder emptying (expressed as percentage of original 99Tc counts in the gallbladder) were measured at intervals for 120 min following feeding. Feeding resulted in prompt gallbladder emptying. Sphincter of Gddi spike burst activity was not altered significantly in the first 30 min after feeding, suggesting that motor activity in the sphincter of Oddi does not initially influence bile flow. Subsequently, spike burst activity increased progressively, suggestingthat sphincter of Oddi motor activity may accelerate bile delivery into the duodenum during later phasesofgallbladder emptying. 0 1988 Academic Press, Inc.
INTRODUCTION
The use of radionuclide imaging to study biliary motility in vivo has been reported in animals and humans [ l-51. Using these techniques it is possible to study the interaction of gallbladder emptying with motor activity of the biliary sphincter of Oddi. It has been postulated [6, 71 that such interactions may regulate the flow of bile into the duodenum following feeding. In this study we used radionuclide imaging techniques to quantitate postprandial gallbladder emptying while monitoring electrical spike burst activity of the sphincter of Oddi in the opossum. The results suggestthat motor activity in the biliary sphincter may not only restrict flow of bile into the duodenum before feeding, but also may accelerate emptying of bile from ’ Supported by NIH Grant 2-RO1-DK3583 l-05. 0022-4804/88 $1.50 Copyri8ht Q 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
the gallbladder into the duodenum during the late postprandial period. METHODS
Studies were carried out in 10 adult opossums (Didelphis virginiana, R-Zoo, Neshkoro, WI) of either sex and parasite free. The animals were anesthetized with sodium pentobarbital (20-30 mg/kg) injected intraperitoneally and maintained with small intravenous pulses as needed. Using sterile surgical technique a midline abdominal incision was made and the biliary tree was exposed, The sphincter of Oddi was identified as a thick smooth muscle structure l-2 cm in length extending from the junction of the common bile duct and pancreatic duct (proximal sphincter) to the duodenal wall (distal sphincter). Bipolar insulated copper wire (0.12 in. in o.d., Biomed Wire, Cooner Co.,
596
PARODI ET AL.: SO AND GALLBLADDER
Chatworth, CA) electrodes were secured to the smooth muscle of the proximal and distal sphincter (Fig. 1). A monopolar shield electrode was placed within the duodenal wall 10 cm distal to the choledochoduodenal junction to serve as a ground. The electrode wires were brought out through the abdominal incision, tunneled subcutaneously to the midscapular region, and brought out through a transcutaneous stainless steel cannula fitted with an Amphenol-five pin connector. The electrodes themselves were constructed by removing a 2-mm band of insulation, 4 cm from the end of the electrode wire. The electrode was introduced into the tissue by threading the wire through a 23-gauge hypodermic needle passed through the circular layer of muscle. The bare section of the wire was positioned within the tissue and the free exiting end was tied to the fixed entering end. The abdominal wall and skin were then closed and a longitudinal incision was made in the right anterolateral neck. The right jugular vein was dissected and a 9-Fr Teflon catheter with a vascular port (Model GPI, Norfolk Medical, Inc., Stokie, IL) was inserted in the vein and was placed in a subcutaneous pocket. After a 2-week recovery period, each animal was fasted overnight and a slow intravenous saline infusion was started (0.5 ml/min)
FIG. 1. Chronic experimental preparation in the opossum. Bipolar wire electrodes are secured to the proximal and distal sphincter of Oddi.
EMPTYING
597
through the vascular port. The animals then received pancuronium bromide (0.1 mg/kg) intravenously, and were intubated endotracheally and ventilated mechanically. Paralysis was maintained with intravenous pulses of pancuronium bromide. Skeletal muscle paralysis was utilized to eliminate motion artifact on the radioimaging recordings. The electrocardiogram of the animal was monitored throughout each experiment. Arterial blood gas analysis was performed intermittently to assure the consistency of mechanical ventilation. The output of the biliary electrode was connected to an eight-channel Gould chart recorder (Gould Electronics Division, Cleveland, OH). The polygraph was calibrated to record slow waves with a low cutoff filter of 0.16 Hz, and spike bursts with a high cutoff filter of 30- 100 Hz. Myoelectric activity of the sphincter of Oddi was recorded continuously using a chart speed of 1 mm/set. After recording two cycles of the interdigestive motor complex (MMC), the abdomen was centered over the gamma camera and 2 mCi of 9gTc-HIDA was infused via the vascular port. The isotope was permitted to concentrate in the gallbladder for 30 min before each experimental protocol was begun. Gamma radiation over the abdomen was measured with a medium energy collimator. Serial analog images were processed every minute by a Techni-Care T60 computer system and stored on a floppy disc. When the gallbladder was well visualized with radioisotope (30 min, in all cases)a 6-mm orogastric tube was placed and a liquid test meal composed of 3.3 g of fat, 3.5 g of carbohydrate and 0.9 g of protein, with a total volume of 30 ml (long chain triglycerides, Sandoz Pharmaceuticals Corp., NJ, and Enrich Source Fiber, Ross Laboratories, Division of Abott Laboratories, Columbus OH), was instilled into the stomach. Radionuclide imaging was continued for 4-6 hr until at least 50% of the radioisotope had disappeared from the gallbladder and appeared in the duodenum. The pancuronium bromide was reversed with neostigmine (0.25 mg intrave-
598
JOURNAL OF SURGICAL RESEARCH: VOL. 44, NO. 5, MAY 1988
nously and repeated as needed) and subcutaneous atropine and the animal was allowed to recover.
0.6-J
i
o[
1 0
I 30
i 60
I 90
! I20
TIME 1min 1 FIG. 3. Time activity curve of 99Tc-HIDA”within the gallbladder in a typical experiment. tJo represents the time required for 50% depletion of gallbladder counts.
Four of the ten opossums were used to determine the effect of pancuronium bromide on sphincter of Oddi myoelectric activity. After an overnight fast, myoelectric activity was recorded for 2 MMC cycles. Intravenous pancuronium was then administered as described above and recordings were continued for 4 to 6 hr, covering at least two further cycles of the MMC. The muscle relaxant was then fully reversed. Myoelectric recordings were visually inspected and the spike bursts per minute were counted and plotted as a histogram. The mean length of the first two MMC cycles and the mean spike bursts per MMC before and after pancuronium were computed and statistically compared using the Student t test for paired data.
ComputerAnalysis of Radioactivity The data were stored on floppy discs at 1-min intervals and anatomic regions of interest, i.e., liver, gallbladder, and duodenum, were outlined on the screen of the computer terminal using a mouse. Counts in each region were plotted on a time activity curve. FIG. 2. Serial scintigram, from the first hour of a typiFrom the curve the percentage of counts cal experiment. Left column represents actual scintiemptied by the gallbladder was calculated grams; right column delineates area of interest selected for measurement of actual counts. Rapid uptake of iso- from the equation: tope by the liver and concentration in the gallbladder in the fasting state are noted (top panel). Animals were fed at 30 min. After feeding, rapid emptying occurs and radionuclide appears within the duodenum.
GB cpm before meal - GB cpm after meal x IO0 GB cpm before meal *
PARODI ET AL.: SO AND GALLBLADDER
EMPTYING
599
TABLE I POSTPRANDIALSPHINCTER OFODDI MYOELECTTRICACTIVITY AND GALLBLADDEREMPTYING Time (mitt) 0
meal
OGBemptied 9.3 + 2.1 PSOspikefrequency (meanSB/IOmitt) 23.3f 3.1 DSOspikefrequency (meanSB/10min) 19.8f 3.7
10
20
30
40
60
50
9.7 2 3.2 21.9+ 4.3*.** 25.4+ 5.9* 31.1+ 6.F
34.0+ 7.v
38.3?I 8.5*
24.6+ 3.4 31.3k 3.0
32.12 2.7 35.5+ 4.1*v** 35.0k 3.1*.*** 31.2+ 2.7
21.4+ 0.3 25.0+ 4.2
25.5k 0.2
30.7f 5.1**
29.9+ 4.8
24.8f 3.7
Nole.Percentage of gallbladderemptyingandsphincterof Oddi mycelectricactivity afterintragastticinstillationof a meal.Values areexpressedasmeans+ SEM. * P < 0.05comparedwith control. l * P < 0.05compared with preceedingtime period. *** P c. 0.05comparedwith first interval afterfeeding.
Analysis of Myoelectric Recordings
shown in Fig. 2. A time activity analysis for a typical experiment is shown in Fig. 3. As illustrated in the figures the marker was rapidly concentrated within the gallbladder, reaching a maximum at 20-30 min after infusion. When the meal was administered a prompt decrease in gallbladder counts was observed. Maximum gallbladder emptying occurred between 10 and 20 min after the meal; by the end of that interval 2 1.9 + 4.3% of gallbladder counts had emptied (n = 10, P < 0.05 compared to before feeding, Table 1).
Recordings of myoelectric activity in the biliary sphincter were inspected and total spike burst frequency within IO-min intervals was analyzed. The data from the gallbladder radionuclide activity curve and sphincter of Oddi spike burst frequencies were entered in a worksheet data base on a Lotus l-2-3 program (Lotus Development Corp., Cambridge MA) on an IBM PC/AT. Statistical analyses were performed using Student’s t test for paired or unpaired data where appropriate. A P level of co.05 was Sphincter of Oddi Myoelectric Activity accepted as significant.
Comparison of fasting myoelectric activity in the presence and absence of intravenous Gallbladder Emptying pancuronium is shown in Table 2. No statistically significant changes were observed in Serial scintigraphs of the abdomen followspike burst frequency and duration of the ing intravenous infusion of 99Tc-HIDA are RESULTS
TABLE 2 EFFECTOFPANCURONIUMBROMIDEON FASTINGMYOELEC~RICACTIVITY OFTHE OPOSSUMSPHINCTEROFODDI Before PSO MMC cycle length (min) DSO MMC cycle length (min) PSO spike bursts/cycle DSO spike bursts/cycle Note. n = 4. ns = not significant.
98.7 +- 6.6 104.3 + 7.3 288.7 + 26.0 223.3 +_22.8
After 91.7 f 104.0 + 263.3 f 209.2 k
5.1 7.9 20.0 26.5
ns ns ns ns
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JOURNAL OF SURGICAL RESEARCH: VOL. 44, NO. 5, MAY 1988
MEAL
Y 4-
iIs ::1-1
+
2
3
4
5
6
7
6
TIME (hours) FIG. 4. Spike burst frequency histogram, based on S-min interval averages for the proximal and distal sphincter of Oddi prior to and after intragastric meal instillation.
MMC in the sphincter of Oddi. Administration of the fat-containing meal disrupted the interdigestive myoelectric cycles and resulted in an overall increase in sphincter spike burst activity (Fig. 4). Figure 5 compares the time course of changes in myoelectric activity of the proximal and distal sphincter with the time course of gallbladder emptying as obtained by radionuclide imaging. Proximal and distal sphincter spike burst activity did not change during the first IO-min interval after feeding, and thereafter, sphincter spike frequency increased but did not reach significance throughout the first 30-min postprandial interval (Table 1). During the next 30min interval, spike burst frequencies increased significantly in conjunction with progressive emptying of 99Tc-HIDA from the gallbladder. Gallbladder emptying was sustained between 60 and 150 min after feeding and resulted in a depletion of more than 50% of counts. Thus, maximum myoelectric activity of the biliary sphincter was observed not during gallbladder filling before feeding, or at the initiation of gallbladder contraction, but well after the phase of the most rapid postprandial gallbladder emptying. DISCUSSION
Technetium-99-HIDA imaging has been used as a sensitive method for evaluating gallbladder function and disease due to its
high uptake and biliary excretion in both animals and humans. It has been demonstrated in humans and dogs that emptying of bile from the gallbladder is in concert with the later phase II of the migrating motor complex of the small intestine [S]. Also, a reflex interaction of the gallbladder, duodenum,
3 LL
01
30
60 so TIME (minutes)
120
J
I?io
FIG. 5. Simultaneous g9Tc-HIDA time activity curve within the gallbladder (top panel) and proximal sphincter of Oddi (PSO) and distal sphincter of Oddi (DSO) spike burst frequencies (bottom panel) observed before and after intragastric meal instillation. (Values are expressed as means + SEM of IO-min intervals for experiments, n = 10.)
PARODI ET AL.: SO AND GALLBLADDER
and sphincter of Oddi has been suggestedin the regulation of sphincter function and gallbladder motility [6]. Similarly, a relationship between gallbladder evacuation and motor activity of the sphincter has been suggested. It has been demonstrated that in the opossum cholecystokinin (CCK) increases both gallbladder emptying and sphincter of Oddi myoelectric spike activity in a dose-dependent fashion [9-131. This is similar to the effect of CCK in the sphincter of Oddi of the prairie dog, rabbit, and, in some cases,man, and is different from the response observed in the cat, guinea pig, and dog [ 121.The relationship between myoelectric activity of the sphincter of Oddi and gallbladder emptying has not been systematically studied. Using the methodology described above we quantitated gallbladder emptying after instillation of a test meal into the stomach. Our results indicated that the gallbladder of the opossum emptied promptly immediately after feeding. Sphincter of Oddi myoelectric activity remained unchanged during this early postprandial period and did not significantly accelerate until 40 min after feeding. These findings suggesta delay in sphincter of Oddi activation compared to the onset of gallbladder emptying. The observations may have been influenced by the skeletal muscle relaxant employed to eliminate motion artifact; however, our studies demonstrated no effect of pancuronium on biliary myoelectric activity. Additionally, pancuronium has been previously shown to have minimal influence on gastrointestinal smooth muscle [14, 151. The delay in increased spike burst activity in the biliary sphincter suggests that early after feeding, in response to enteric content, bile flow into the duodenum is largely dependent upon contraction of the gallbladder with the sphincter of Oddi playing a minor role in modulating bile flow. In the late postprandial period, as gallbladder emptying slows and overall bile flow declines, sphincter motility increases. During this period the sphincter may serve to actively pro-
EMPTYING
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pulse bile into the duodenum. Alternatively, the sphincter may simply be responding secondarily to increased postprandial biliary flow and pressure, serving as a meter or valve. Data supporting both hypotheses in many animal species, including man, exist [9, 10, 16-l 81. Further studies monitoring gallbladder and sphincter of Oddi myoelectric activities, bile flow, and biliary pressure are warranted. REFERENCES 1. Engler-t, E., and Chiu V. Quantitative analysis of human biliary evacuation with radioisotopic technique. Gastroenterology 50: 506, 1966. 2. Harvey, E., Loberg, M., Ryan, J., et al. Hepatic clearance mechanism of Tc-99-HIDA and its effect on quantitation of hepatobiliary function. J. Nucl. Med. 20: 310,1979. 3. Ivy, A. C., and Bergh, G. A. The applied physiology of the extrahepatic biliary tract. JAMA 103: 2500,
1934. 4. Krishnamurthy, G. T., Bobba, V. R., and Kingston,
E. Radionuclide ejection fraction: A technique for quantitative analysis of motor function of the human gallbladder. Gastroenterology 80: 482, 1981. 5. Wistow, B. W., Subramanian, G., Vanheeertum, R. I., et al. An evaluation of Tc-99m-labeled hepatobiliary agents. J. Nucl. Med. 18: 455, 1977. 6. Muller, E. L., Lewinski, M. A., and Pitt, H. A. The cholecysto-sphincter of Oddi reflex. J. Surg. Rex 36: 377,1984. I. Takahashi, I., Kern, M. K., Dodds, W. J., Hogan, W. J., Sama, S. K., Soergel, K. H., and Itoh, Z. Contraction pattern of opossum gallbladder during fasting and after feeding. Amer. J. Physiol. 250: G227, 1986. 8. Itoh, Z., and Takahashi, I. Periodic contractions of
the canine gallbladder during the interdigestive state. Amer. J. Physiol. 240: G 183, 1981. 9. Becker, J. M., Moody, F. G., and Zinsmeister, A. R. Effects of gastrointestinal hormones on the biliary sphincter of Oddi of the Opossum. Gastroenterology 82:1300,1982.
10. Behar, J., and Biancani, P. Effect of cholecystokinin and the octapeptide of cholecystokinin on the feline sphincter of Oddi and gallbladder. J. Clin. Invest. 66:1231,1980.
11. Behar, J., and Biancani, P. Pharmacologic characterization of excitatory and inhibitory cholecystokinin receptors of the cat gallbladder and sphincter of Oddi. Gastroenterology 92: 764, 1987.
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12. Sarles, J. C. Hormonal control of sphincter of Oddi. Dig. Dis. Sci. 31: 208, 1986. 13. Spellman, S. J., Shaffer, E. A., and Rosenthal, L. Gallbladder emptying in response to cholecystokinin. A cholescintigraphic study. Gastroenterology 7: 115, 1975. 14. Bromage, P. R. Pulmonary complications of regional anesthesiafor obstetrics. In S. M. Shnider and G. Levinson (Eds.), Anesthesia for Obstetrics, 2nd ed. Baltimore: Williams & Wilkins, 1987. 15. Scott, R., and Savarese, J. The cardiovascular and autonomic effects of neuromuscular blocking agents. In R. L. Katz (Ed.), Muscle Relaxants: Basic
and Clinical Aspects. New York: Grune & Stratton, 1985. 16. Ashkin, J. R., Lyon, D. T., Shuli, S. C., Wagner, C. I., and Soloway, R. D. Factors affecting delivery of bile to the duodenum in man. Gastroenterology 74: 560, 1978. 17. Scott, G. W., Smallwood, R. E., and Rowlands, S. Flow through the bile duct after cholecystectomy. Surg. Gynecol. Obstet. 240: 912, 1975. 18. Tansy, M. F., Innes, D. L., Martin, J. S., and Kendall, F. M. A functional description of canine choledochoduodenal flutter valve. Amer. J. Dig. Dis. 21: 223, 1976.