Interdigestive Gallbladder Bile Concentration in Relation to Periodic Contraction of Gallbladder in the Dog

Transcription

1 GASTROENTEROLOGY 1982;83: LIVER AND BILIARY TRACT Interdigestive Gallbladder Bile Conentration in Relation to Periodi Contration of Gallbladder in the Dog ZEN ITOH, ITSUO TAKAHASHI, MIT SUO NAKA Y A, TOMOAKI SUZUKI, HISAKO ARAI, and KATSUMI WAKABAYASHI Gastrointestinal Laboratories. Department of Surgery. Gunma University Shool of Mediine. and Hormone Assay Center. Institute of Endorinology. Gunma University. Maebashi. Japan Interdigestive hanges in the onentration of bilirubin and sodium in gallbladder bile olleted through a hronially indwelling tube in the gallbladder were measured in onjuntion with periodi ontrations of the gallbladder and immunoreative motilin onentration in plasma in onsious dogs. It was found that the bilirubin onentration in the gallbladder bile inreased as stepped ars that show periodi dereases with eah yle of the interdigestive migrating ontration in the stomah. The yle for bilirubin onentration was then repeated. with higher maximum bilirubin values ourring during suessive interdigestive migrating ontrations yles. In ontrast, the sodium onentration did not progressively inrease but flutuated widely in assoiation with hanges in the bilirubin onentration. Peaks in the onentration of bilirubin and sodium generally oinided and ourred in the phase 3 period of the interdigestive migrating ontrations in the stomah and the duodenum. However. the initial rise in the sodium onentration after the fall took plae earlier than that in the bilirubin onentration in almost one-half of all experiments. Peaks of plasma immunoreative motilin onentration oinided with the maximum onentration of gallbladder bile in eah of the onentrating yles. but there was no orrelation between the two when ompared over the entire period of the experiments. These findings suggest that the interdigestive periodi ontrations in the gallbladder may playa role in the progressive onentration of bilirubin in gall- Reeived Otober Aepted April Address requests for reprints to: Zen Hoh. M.D.. Ph.D. GI Laboratories. Department of Surgery. Gunma University Shool of Mediine. Maebashi. Japan 371. This work was supported in part by a grant-in-aid from the Ministry of Eduation of Japan to Zen Hoh by the Amerian Gastroenterologial Assoiation bladder bile; that is, gallbladder relaxation after termination of the immunoreative motilin onentration may be assoiated with aspiration by the gallbladder of dilute hepati bile. This may in turn stimulate further gallbladder reabsorption. These studies do not allow firm onlusions to be drawn regarding the role of motilin in gallbladder absorption. It is a well-known physiologi fat that the major funtion of the gallbladder is to onentrate hepati bile and to store it between meals, and that when food enters the duodenum the onentrated bile is emptied into the duodenum. Emptying of the gallbladder bile into the duodenum is ahieved by gallbladder ontration indued by holeystokinin (CCK) released from the duodenum and the upper jejunum in response to food and its digesta (1). On the other hand, we have reently observed periodi ontrations of the gallbladder whih our in lose assoiation with the inter digestive migrating ontrations (IMC) in the stomah and whih an be reprodued by exogenous administration of motilin (8). It is not yet known, however, whether or not these inter digestive gallbladder ontrations have any physiologi signifiane in the inter digestive funtion of this organ. In these experiments, we have studied hanges in the onentration of bilirubin and sodium in gallbladder bile during the inter digestive state and the relationship of these hanges to the periodi ontration and plasma motilin onentration. Materials and Methods Preparation of Animals Four healthy adult mongrel dogs of both sexes weighing 15-2 kg were used in the present study. Under n.... ~ n o1 t... = Jnnne. A : n " ~ f~ l n ~

2 646 ITOH ET AL. GASTROENTEROLOGY Vol. 83, No. 3 general anesthesia by Lv. injetion of pentobarbital sodium (3 mg/kg body wt), the abdominal avity was opened by a midline inision. A fore transduer (7) onstruted in this laboratory was sutured on the serosa of the body of the gallbladder with a 4- nontraumati needle to measure irular musle ontration. The lead wires from this transduer were direted to the hepati hilum along the ysti dut and taken out into the open abdominal avity through the dependent interlobular spae of the liver. These proedures minimized the number of artifats on the transduer due to respiration and movement of animals. A Silasti tube (62-25, Dow Corning, Midland, Mih.) was introdued into the gallbladder through an inision made on the fundus of the gallbladder and was held in plae by a purse string suture and was alled the "gallbladder tube." Two other fore transduers were also sutured onto the serosa of the gastri antrum 3 m proximal to the pylori ring and of the mid-duodenum just opposite to the opening of the main panreati dut to measure irular musle ontration at eah site. Lead wires of the fore transduers were taken out of the abdominal avity through a stab wound made on the left abdominal wall and pulled out of a skin inision made between the sapulas through a subutaneous tunnel on the left ostal flank. The lead wires were sutured with silk onto the skin adjaent to the skin inision. The gallbladder tube was brought from the abdominal avity and fixed onto the skin adjaent to the laparotomy wound. The gallbladder tube was stopped with a tiny plasti plug to prevent leakage of bile. After the abdominal surgery, a Silasti tube (62-25, Dow Corning) was introdued into the superior vena ava through a branh vein of the external jugular vein and the outer end of this tube also was fixed onto the nek skin with silk. After filled with heparinized saline, the tube was also tipped with a tiny plasti plug and used as a route for postoperative fluid infusion, and withdrawal of blood samples for measurement of plasma motilin onentration. After the operation a jaket protetor of anvas was plaed on the dog to protet the lead wires and Silasti tubes from damage due to the dogs' srathing. The dogs were fasted for 3 days postoperatively and maintained by Lv. infusion of latate Ringer's solution (6 ml/kg body wt). During the experimental period, they were fed one a day with a measured volume of dog food (Gaines Meal, dry type, 2 g/kg body wt) at a fixed time and given water ad libitum. Experimental Proedures Experiments were arried out during the period from the 3rd to 6th week after the operation. Dogs were housed in experimental ages. Conneting ables from amplifiers, whih were hung from the eiling by an elasti ord just above the dog, were firmly fixed onto the dog's protetor and then onneted to the lead wires from the transduers with a small onnetor under the protetor. Contratile hanges in the gallbladder, the stomah, and the duodenum were ontinuously reorded on a penwriting reorder at a paper speed of 1 mmlmin for general inspetion of the ontratile pattern in those three sites. Detailed hanges were reorded on another pen-writing reorder, whih was run at a paper speed of 1-13 mml min. For approximately 4 h after the first IMC was onfirmed to our in the stomah, samples of bile and blood were olleted at 15-min intervals. Care was taken to avoid ontaminating the multiple samples of blood and bile. Corresponding to a dead spae in the gallbladder tube,.3 ml of bile was aspirated and then.2 ml was olleted for measurement, after whih the first.3 ml was returned to the gallbladder to minimize the effet of aspiration. Aliquots of bile,.1 ml, were diluted 11 times with redistilled water for measurement of bilirubin onentration in a spetrophotometer (UV-14, Shimadzu Seisakusho Ltd., Kyoto, Japan) at a wave length of 46 nm. A standard urve for bilirubin was onstruted by using bovine bilirubin (Sigma Chemial Co., St Louis, Mo.) dissolved in benzene. The remaining aliquots of bile were diluted to 1 times with redistilled water and the sodium onentration was determined in a flame photometer (25D, Hitahi Seisakusho, Tokyo, Japan). Experiments were repeated at least five times at 3-5-day intervals on eah of the 4 dogs. In three experiments involving measurement of the bile onentration, blood samples were olleted together with bile aspiration for measurement of the plasma immunoreative motilin (IRM) onentration. Blood was olleted into hilled tubes with Trasylol (1, KU/ml, Bayer Japan, Tokyo, Japan) 111 volume of the blood samples. The samples were mixed and entrifuged at 3 rpm for 5 min at O C, and the supernatants were stored at -3 C until radioimmunoassay for motilin. Details of the proedures for radioimmunoassay for motilin have been reported previously (9). Data obtained in the present study were analyzed statistially with a Student's t-test and p-values <.5 were onsidered signifiantly different between paired data. Results were presented in the form of the mean ± SE of the mean for eah of the 4 dogs, but the mean values for the 4 dogs were shown as the mean of the mean values for eah of 4 dogs. Results Interdigestive Changes in Bilirubin and Sodium Conentration in the Gallbladder and Immunoreative Motilin Conentration in Plasma Figure 1 shows 5-h atual hanges during the interdigestive state in a dog. The IMC ourred at onstant intervals in the gastri antrum and the duodenum. In assoiation with the initial phase of these ontrations in the duodenum, toni ontrations of the gallbladder were seen to our and return to the basal level before the gastri and duodenal IMC reah phase 3 ativity. The bilirubin onentration was, on the whole, inreasing during the interdigestive state, but the inrease progressed as a series of stepped ars that show periodi de-

3 September 1982 INTERDIGESTIVE GALLBLADDER FUNCTION :u C <II u C u ~ I E ::J V'l :;: ~ i <II u E "CI I a a. I U a:: ! 6 ~ 6 4 /....(i ~... -._. 2 a" Gastri Antrum Duodenum I i I \ /. d b. _. o - ' ~ 3 25.Q 2 t 1i ::J 15.!:: i:o 1 ~ 1 U E Gall bladder Time Intervals, 1 hr Figure 1. Interdigestive 5-h hanges in gallbladder bilirubin and sodium onentration (top). plasma IRM onentration (middle), and ontratile ativity in the gastri antrum. the duodenum. and the gallbladder (bottom) in a onsious dog. Three shaded vertial1ines indiate simultaneous ourrene of onentration and motor events. reases (15.1 ±.74 mg/dl) paralleling the IMC yles. The yle for bilirubin onentration was then repeated. with a higher maximum bilirubin value ourring in eah suessive IMC yle. Peaks of bilirubin onentration in eah onentration yle usually oinided with the phase 3 period of the IMC in the stomah and the duodenum as shown by the shaded line in Figure 1. Consequently. when the gastri and d u o d IMC ~ n were a l terminated. the bilirubin onentration began to derease and then in approximately 3 min it started to inrease again and during the subsequent IMC period reahed a higher onentration than before. On the other hand, the sodium onentration in the gallbladder did not progressively inrease but flutuated widely with the yle of the IMC. With the fall in bilirubin onentration, the sodium onentration dereased muh more sharply than the bilirubin onentration and reahed the bottom in 15 min, as seen in this figure. Initiation of the derease in the onentration of both sodium and bilirubin usually took plae at the same time. In the seond IMC period in Figure 1, however, it is shown that the sodium onentration dereased 15 min later than bilirubin, but suh a dissoiation was rather exeptional and ourred in only 6.3% of all the measurements. On the ontrary, the inrease in sodium onentration ourred earlier than that of bilirubin, as seen in the first and seond IMC period in Figure 1. This dissoiation was observed in 42% of all experiments. The sodium onentration inreased thereafter, but it did not exeed a level of approximately 35 meq/l. In Figure 2, these findings are shematially illustrated to make it easier to understand the terminology used in the present study as well as the orrelation between the gallbladder bilirubin onentration and motor ativity in the stomah and the gallbladder. During the interdigestive state, the plasma IRM onentration flutuated as shown in Figure 1, and its peaks oinided with the gastri IMC, as reported previously (9). Peaks of plasma IRM onentration also orresponded to those of the bilirubin and sodium onentration in the gallbladder bile as indi-

4 648 ITOH ET AL. GASTROENTEROLOGY Vol. 83, No.3 I-----on:entrating yle gastri motility gallbladder motility peak Figure 2. Shemati presentation of the present findings on the relationship between the gallbladder bile onentration and motor ativity in the stomah and the gallbladder. This shema also illustrates speifi terminology used in the present study to indiate levels and yles of bile onentration in relation to motor yles. ated by the shaded vertial lines in Figure 1. Changes in plasma IRM, however, were not parallel with those of gallbladder bile onentration when ompared over the entire period of the experiments, as seen in this figure; during the quiesent periods in the interdigestive state, the plasma IRM onentration was dereasing but the onentration of sodium and bilirubin was inreasing, partiularly during the latter half of the quiesent period. Detailed Changes in Bilirubin Conentration in the Gallbladder Figure 3 shows the results of five experiments in whih the bilirubin onentration in the gallbladder bile in eah of 2 dogs was measured. Open irles on eah urve in the figure represent the period of IMC in the stomah and the duodenum. As seen in this figure, the bilirubin onentration in the gallbladder bile inreased progressively as a series of stepped ars that orrelated with the IMC yles. The initiation of dereases in the bilirubin onentration oinided with the termination of eah IMC in the stomah and the rate of oinidene in dog 1 was 68%, in dog 2 66%, in dog 3 81 %, and in dog 4 73%. 1:11 general, the bilirubin onentration inreased in a stepwise fashion in lose assoiation with the IMC in the stomah and the duodenum. The inrease in the bilirubin onentration in the first 15 min (17.6 ± 1.49 mg/dl) was greater than in the last 15-min period (7.4 ±.75 mg/dl) in the onentrating yles (note the terminology shown in Figure 2). It was also found that the bilirubin onentration in gallbladder bile, though it varied between individual dogs and periods during the interdigestive state, inreased with time during this period. The inrease in the onentration of bilirubin in eah of the onentrating yles (peak to peak) was found to be 49.4 ± 2.77 mg/dl as s4wn in Table 1. However, the mean derease in the bilirubin onentration after the gastri IMC (peak to bottom) was 15.1 ±.74 mg/dl. The mean lifferene between the lowest and highest onentration (bottom to peak) in the onentrating yles was 59.8 ± 1.18 mg/dl so that the average rate of onentration during the inter digestive state was mg/dl min. Relation to Motilin The rate of hange in the onentration of the gallbladder bile in every 15 min was ompared with " -1 2 E.2... r!! ~ (1) 4 u u : 35 :J... id Time intervals, 3min Figure 3. Interdigestive hanges in gallbladder bilirubin onentration in 5 experiments in 2 onsious dogs. Open irles on the urves represent periods of interdigestive ontrations in the gastri antrum.

5 September 1982 INTERDIGESTIVE GALLBLADDER FUNCTION 649 Table 1. Conentration and Dilution of Gallbladder Bilirubin and Sodium During the Interdigestive Conentrating Cyles Bilirubin Sodium Conentration Dilution Conentration Dilution Dog No. peak to peak bottom to peak peak to bottom bottom to peak peak to bottom ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 5.4 Mean 49.4 ± ± ± ± ± 4.6 Values are mean ± SE of the mean in mg/dl for bilirubin and in meq/l for sodium of the means obtained from 1 observations for individual dogs. Mean ± SE of the means obtained from individual mean values. the orresponding plasma IRM onentration in eah of the 4 dogs, the results of whih are shown in Table 2. It is ertain that at peaks of plasma IRM onentration, the bilirubin onentration was maximum but there was no orrelation between the hanges in plasma IRM and bilirubin onentration when ompared over the entire period of the experiments, as shown in Table 2. Disussion In the present study, we aimed at finding out the physiologial signifiane of the periodi on trations of the gallbladder, whih we reently found to exist during the inter digestive state (8). Measurements in gallbladder bile during the interdigestive state revealed that bilirubin onentration progressively inreased, but that its rise was in a stepwise fashion: a distint derease in gallbladder bile onentration followed by a sharp rise was observed and orrelated with episodes of periodi ontrations of the gallbladder. On the other hand, the sodium onentration did not progressively inrease but flutuated widely with the IMC yles. In assoiation with the derease in the bilirubin onentration, the sodium onentration dropped to the bottom level in 15 min muh more sharply than the biliru- Table 2. Fifteen-Minute Conentrating Rate of Gallbladder Bilirubin and Plasma Immunoreative Motilin Level During the Interdigestive State Coeffiient orrelation between 15-min hanges in gallbladder bilirubin onentration Dog and p-values No. absolute IRM values I5-min hanges in IRM < (n = 22) (n = 22) (n = 23).149 (n = 23) (n = 27).2237 (n = 27) (n = 19).215 (n = 19).4555 Values are oeffiient orrelation obtained from 3 experiments and numbers in parentheses are number of measurements. bin onentration did and soon started to inrease again. However, it eased at approximately 35 meq/l toward the end of eah onentration yle: this may mean that the gallbladder bile is saturated in terms of sodium onentration and a further onentration of bile is impossible beyond this onentration of sodium at approximately 35 meq/l. In the present study, we onfirmed that gallbladder bile is onentrated during the interdigestive state as stated in textbooks of gastroenterology. However, the interdigestive periodi dilution of gallbladder bile during the bile onentrating proess was quite a new finding and the fat that the periodi dilution is losely orrelated with the yles of the periodi ontration of the gallbladder appears to provide us with a key to the mehanism of this finding. The dilution of gallbladder bile implies an inflow of dilute bile into the gallbladder from the outside, whih may at first be interpreted as a result of relaxation of the ontrated gallbladder. In order for the gallbladder one ontrated to return to its preontration level, it is theoretially impossible to return to its original level without aspirating a volume of bile equal to that whih had been emptied by the ontration. Therefore, it is onluded that a fairly large volume of hepati bile is delivered to the gallbladder, judging from the rapid derease in sodium onentration in 15 min. Another possible explanation for dilution of gallbladder bile ould be seretion of water from the gallbladder. While this possibility annot be rigorously exluded, it seems a less likely explanation for the present findings. As to the rapid inrease in the bilirubin onentration observed after the dilution of bile, it may be onluded that absorption rate of water and eletrolytes is greater in dilute bile than in onentrated bile (6). A more important but a diffiult problem onernihg the dilution-onentration proess in the gallbladder is the dissoiation of the initiation of onentration of sodium and bilirubin. Initiation of the rise in sodium onentration took plae earlier than

6 65 ITOH ET AL. GASTROENTEROLOGY Vol. 83, No. 3 that of the bilirubin onentration in approximately 4% of all the observations in the present study. This is a puzzling phenomenon: while the bilirubin onentration was still dereasing, the sodium onentration had already started inreasing as seen after the termination of the first and seond IMCs in Figure 1. It is diffiult to oneive of a simple dilution-onentration mehanism to explain these nonparallel hanges in the two solutes in gallbladder bile. If absorption of sodium from the gallbladder muus membrane is extremely rapid, ould suh a dissoiation our between the two sine bilirubin is not absorbed from the gallbladder? This problem will have to be dealt with in future studies. On the other hand, the periodi ontration of the gallbladder is likely to disharge a part of the gallbladder bile into the ommon bile dut or into the duodenum. Or, are the periodi ontrations aused by a redution in the intragallbladder volume due to absorption? In this respet, reent reports support our findings. Keane et a1. (14) and DiMagno et a1. (4) studied the relationship between fasting panreati and biliary seretion and duodenal phase 3 ativity in the dog and desribed the assoiated inrease in panreati and biliary seretion with intense motor ativity of the duodenum. On the other hand, Peeters et a1. (16) studied normal humans and holeystetomized patients and onluded that the bile aid output into the duodenum during the inter digestive phase flutuates in aordane with the interdigestive motor omplex. The inrease in bile aid output in their report is therefore asribed to an inreased flow of hepati bile. In the study of water absorption in the gallbladder in relation to gut hormones, we ould not larify whether or not motilin is involved in the mehanism of gallbladder bile onentration in the present study but a series of studies on gut hormones have been reported by Svanvik and Jansson. They studied net water absorption in the perfused gallbladder of the at anesthetized with hloralose in reponse to various gut hormones (17). These authors found that seretin (13) and vasoative intestinal polypeptide (VIP) (14) inhibited the net water absorption while gluagon (12), CCK (13), or gastri inhibitory polypeptide (GIP) (12) did not Signifiantly affet the onentrating funtion in the gallbladder. In addition, Jansson (1) studied the absorption funtion in the gailbladder during the aid and alkaline infusion into the duodenum in anesthetized ats and found that it redued the net water absorption by duodenal aidifiation, but aused no hanges in gallbladder net water transport or motility in the gallbladder due to alkaline infusion. Under the anesthetized ondition, the true physiologi effet of motilin is not manifested even though alkalinization stimulates motilin release (7). In the searh for literature desribing water absorption in the gallbladder, there are no reports of studies on the existene of hormones or hemials whih inrease the net water absorption in the gallbladder. Most of agents or hormones were found to have no signifiant effet on or to inhibit ative and passive ioni transport (2,3). Beause it was demonstrated that gallbladder bile onentration is exaggerated during the interdigestive state, it is reasonable to investigate the effet of motilin on onentrating ativity of the gallbladder beause it is the only gut hormone known to be released during the interdigestive state (9,15) and it mimis the periodi ontration of the gallbladder (8). However, as far as the relationship between hanges in plasma IRM and gallbladder bilirubin onentration is onerned, there was no evidene indiating that the interdigestive bilirubin onentration is ontrolled by the plasma IRM onentration. In onlusion, we onfirmed a vigorous gallbladder bile onentrating ativity during the interdigestive state and the onentration was found to progress in a stepwise fashion with dereases in bilirubin and sodium onentration. The stepwise inrease was losely orrelated with the periodi ontration of the gallbladder and onsequently with those of the IMC in' the stomah and duodenum. Cyli dilution of the sodium onentration in the gallbladder bile is likeiy to playa role in reviving water absorption from the gallbladder muus membrane by diluting the sodium-saturated bile. The ontrol mehanism of the dilution and onentration proesses in the gallbladder observed during the interdigestive state is not known. Referenes 1. Banfield WJ. Physiology of the gallbladder. Gastroenterology 1975;69: Diamond JM. The absorptive funtion of the gallbladder. J Physiol (Lond) 1962;161: Dietshy JM. Water and solute movement aross the wall of the everted rabbit gall bladder. Gastroenterology 1964;47: DiMagno EP, Hendriks JC, Go VLW, et al. Relationships among anine fasting panreati and biliary seretions, panreati dut pressure, and duodenal phase III ativity Boldyreff revisited. Dig Dis Si 1979;24: Dryburgh JR, Brown JC. Radioimmunoassay for motilin. Gastroenterology 1975;68: Grim E, Smith GA. Water flux rates aross dog gallbladder wall. Am J PhysioI 1957;191: ltoh Z, Honda R, Takeuhi S, et al. An extraluminal fore transduer for reording ontratile ativity of the gastrointestinal smooth musle in the onsious dogs: its onstrution and implantation. Gastroenterol Jpn 1977;12:

7 September 1982 INTERDIGESTIVE GALLBLADDER FUNCTION Itoh Z, Takahashi 1. Periodi ontrations of the anine gallbladder during the interdigestive state. Am J Physiol (Gastrointest Liver Physiol 3) 1981;24:G Itoh Z, Takeuhi S, Aizawa I, et al. Changes in plasma motilin onentration and gastrointestinal ontratile ativity in onsious dogs. Am J Dig Dis 1978;23: Jansson R. Effet of intraduodenal aid on gallbladder net water absorption and motility in the at. Sand J Gastroenterol1978;13: Jansson R, Steen G, Svanvik J. Effets of intravenous vasoative intestinal peptide (VIP) on gallbladder funtion in the at. Gastroenterology 1978;75: Jansson R, Steen G, Svanvik J. A omparison of gluagon, gastri inhibitory peptide, and seretin on gallbladder funtion, formation of bile, and panreati seretion in the at. Sand J Gastroenterol1978;13: Jansson R, Svanvik J. Effets of intravenous seretin and holeystokinin on gallbladder net water absorption and motility in the at. Gastroenterology 1977;72: Keane FB, DiMagno EP, Dozios RR, et al. Relationships among anine interdigestive exorine panreati and biliary flow, duodenal motor ativity, plasma panreati polypeptide and motilin. Gastroenterology 198;78: Lee KY, Chey WY, Tai H-H, et al. Radioimmunoassay for motilin. Validation and studies on the relationship between plasma motilin and interdigestive myoeletri ativity of the duodenum of dog. Am J Dig Dis 1978;23: Peeters TL, Vantrappen G, Janssens J. Bile aid output and the interdigestive migrating omplex in normals and in holeystetomized patients. Gastroenterology 198;79: SvanvikJ, Jansson R. An experimental method for studying in vivo gallbladder absorption. Gastroenterology 1977;72:634-8.