Digestive Diseases and Sciences, Vol. 37, No. 8 (August 1992), pp. 1232-1235
Effect of Cholecystectomy on Gallbladder Bile Composition STEVEN A. AHRENDT, MD, THOMAS H. MAGNUSON, MD, HENRY A. PITT, MD, and KEITH D. LILLEMOE, MD
Human bile samples are commonly used in biliary research; however, the optimal sampling technique is not known. The current study examines whether bile obtained prior to operative manipulation of the gallbladder differs in composition from samples obtained after cholecystectomy. Gallbladder bile samples were obtained from 26 patients undergoing cholecystectomy. An initial sample was obtained prior to manipulation or devascularization of the gallbladder, and a second sample was obtained after the removal of the gallbladder from the operative field. Gallbladder bile pH and total protein were significantly increased in the postcholecystectomy samples. Bile obtained after cholecystectomy also contained significantly less phospholipid. Gallbladder bile cholesterol, total bile acids, bilirubin, ionized and total calcium, cholesterol saturation index, and total lipids were similar between techniques. These results indicate that manipulation of the gallbladder during cholecystectomy produces alterations in gallbladder bile composition. These results also emphasize the need for consistent sampling technique when obtaining samples for biliary research. KEY WORDS: cholecystectomy; bile; bile composition; bile protein; bile pH.
The analysis of gallbladder bile provides essential information in studies of the pathogenesis of gallstones. Obtaining adequate human bile samples is an important step in the experimental process. Gallbladder bile may be obtained by direct percutaneous aspiration, via endoscopic aspiration of bile-rich duodenal fluid, or from the gallbladder at laparotomy (1, 2). Of these three methods only intraoperative sampling produces representative, minimally contaminated samples without additional risk to the patient or study subject. Ideally, intraoperative bile sampling should be performed prior Manuscript received September 16, 1991; revised manuscript received February 7, 1992; accepted February 27, 1992. From the Department of Surgery, The Johns Hopkins Medical Institutions, Baltimore, Maryland. Supported in part by N.I.H. Grant R29-DK41889 from the National Institutes of Diabetes, Digestive, and Kidney Diseases (K.D.L.) and a Veterans AdministrationMerit Review (H.A.P.). Address for reprint requests: Dr. Keith D. Lillemoe, The Johns Hopkins Hospital, 600 N. Wolfe Street, Blalock 656, Baltimore, Maryland 21205.
1232
to manipulation of the gallbladder, which may introduce blood and cellular debris into the bile (1). Moreover, sampling after cholecystectomy results in the collection of bile after the gallbladder has been rendered ischemic from ligation of the cystic artery. Yet due to technical considerations of surgery, bile samples prior to manipulation may be difficult to obtain. In addition, incomplete aspiration of the in situ gallbladder may not produce a representative sample due to the stratification of bile within the gallbladder (3, 4). Despite these theoretical objections to using samples obtained after operative manipulation of the gallbladder, few data exist comparing bile obtained before with bile obtained after cholecystectomy. The present study was undertaken to determine whether gallbladder bile obtained prior to operative manipulation of the gallbladder was different in biochemical composition from samples obtained after removal of the gallbladder from the operative field. Digestive Diseases and Sciences, Vol. 37, No. 8 (August 1992)
0163-2116D2g0800-1232506.50/09 1992PlenumPublishingCorporation
EFFECT OF CHOLECYSTECTOMY ON BILE COMPOSITION TABLE 1. GALLBLADDERBILE COMPOSITIONBEFOREAND AFTEROPERATIVE MANIPULATIONOF GALLBLADDER Precholecystectomy
pH Total protein (mg/ml) Total bile acids (mM) Total bile acids (molar %) Phospholipids (mM) Phospholipids (molar %) Cholesterol (mM) Cholesterol (molar %) Cholesterol saturation index Total lipids (g/dl) Total bilirubin (~M) Unconjugated bilirubin (~M) Bilirubin monoglucuronide (I~M) Bilirubin diglucuronide (I~M) Ionized calcium (mM) Total calcium (mM)
Postcholecystectomy
6.94 --- 0.07 9.5 --- 2.4 122 --- 18 70.7 -+ 1.6 39 +- 6 21.7 --- 1.3 9.6 -+ 1.3 7.7 1.5 1.42 +- 0.36 12.7 --- 1.7 1930 --- 340 52.7 -+ 17 482 --- 95 1400 --- 250 1.90 -+ 0.23 7.58 -+ 1.0
7.14 --+ 17.4 --113 --72.3 30 19.9 --9.2 +7.7 +1.56 --11.4
-+
0.06a* 3.2b 18 1.2 4b 0.8 1.3 0.8 0.35 1.6
1600 - 290 64.5 -+ 17 391 --- 98 1140 --- 210 1.66 --+ 0.19 7.12 4- 0.9
*a, P < 0.001, and b, P < 0.01 versus precholecystectomy.
M A T E R I A L S AND M E T H O D S Bile samples were obtained from 26 patients undergoing elective, open cholecystectomy. Twenty-two patients had symptomatic gallstones (18 cholesterol and four pigment). The remaining four patients underwent cholecystectomy associated with other hepatobiliary procedures and did not have gallstones. In all patients an initial bile sample was obtained prior to placing clamps on the gallbladder or proceeding with the dissection. The gallbladder was initially manually manipulated in an attempt to mix the gallbladder contents. A 16-gauge needle was inserted into the gallbladder fundus, and 5 ml of bile were anerobically aspirated. Standard cholecystectomy was then performed. Following cholecystectomy the gallbladder was removed from the operative field, and the remaining gallbladder bile was completely aspirated. All bile samples were light-protected and immediately analyzed for ionized calcium and pH using an ion specific electrode (534 Ca2+/pH analyzer, Ciba-Corning, Medfield, Massachusetts). The remaining bile was then centrifuged for 5 min at 2000 rpm and the supernatant stored at - 2 0 ~ C. An aliquot of supernatant (75 ~1) was stored separately with ascorbic acid (1:1 v/v) in sealed light-protected vials for the later determination of bilirubin. After complete bile aspiration, the gallbladder was opened and representative gallstones selected for analysis. A spun bile sediment from both bile samples was examined under the polarizing light microscope for evidence of calcium bilirubinate granules or cholesterol crystals. All bile samples were subsequently rewarmed to room temperature and analyzed for total protein, biliary lipids, bilirubin, and total calcium. Total biliary protein was determined using the Bio-Rad assay (5). Phospholipids were quantitated using the method of Dryer et al (6). Cholesterol concentration was determined using the method of Roschlau et al (7). Total bile acids were measured using the technique of Talalay (8). The cholesterol saturation index was calculated using Carey's critical tables (9). Conjugated, unconjugated, and total billDigestive Diseases and Sciences, Vol. 37, No. 8 (August 1992)
rubin were measured by high-performance liquid chromatography as described by Spivak et al (10). Biliary total calcium was determined spectrophotometrically. Data are presented as the mean +-- SEM. Statistical analysis was performed using Student's t test (two-tailed) for paired data.
RESULTS Gallbladder bile c o m p o s i t i o n in the precholecyst e c t o m y and p o s t c h o l e c y s t e c t o m y samples are presented in Table 1. Biliary p H in the samples prior to any gallbladder manipulation was significantly less than the p H in samples obtained immediately after c h o l e c y s t e c t o m y (P < 0.001). This trend was present in 20 of the 24 samples in which the p H was measured. Biliary protein concentration was markedly increased in samples obtained after r e m o v a l of the gallbladder w h e n c o m p a r e d to samples prior to c h o l e c y s t e c t o m y (P < 0.01). Gallbladder bile obtained after operative manipulation contained significantly (P < 0.01) less phospholipid than bile obtained prior to c h o l e c y s t e c t o m y . Bile cholesterol concentrations and total bile acid concentrations were not affected b y the timing of the sample. The molar percent phospholipid, cholesterol, and bile acid and the ratio o f bile acids to phospholipids were not significantly altered b y the timing o f the bile sample. In addition, the cholesterol saturation index and the total lipid concentration were not affected b y the o p e r a t i v e handling of the gallbladder. Bilirubin monoglucuronide, bilirubin diglucuronide, unconjugated bilirubin, and total bilirubin w e r e similar b e t w e e n the p r e c h o l e c y s t e c t o m y and
1233
AHRENDTET AL postcholecystectomy samples. Operative handling and devascularization likewise did not affect ionized or total calcium concentrations. Microscopic examination of the spun bile sediment in 21 patients revealed cholesterol crystals in three patients, calcium bilirubinate granules in four patients, and both cholesterol monohydrate and calcium bilirubinate crystals in nine patients. In only one of the patients examined did the microscopic exam differ between the two samples. This patient had calcium bilirubinate granules in the sample obtained after cholecystectomy but not in the sample obtained prior to handling the gallbladder. DISCUSSION Accurate, representative bile sampling is crucial in conducting valid research in gallstone pathogenesis. The present study compares two commonly used techniques of handling intraoperative gallbladder bile samples to determine if differences in biochemical composition or microscopic analysis exist between bile obtained prior to handling the gallbladder and bile obtained after cholecystectomy. The results demonstrate that biliary cholesterol, total bile acids, and total and ionized calcium were similar between the pre- and postcholecystectomy samples. In addition, the cholesterol saturation index was not significantly affected by the sampling technique. However, biliary pH, total protein, and phospholipid concentrations did differ between the two groups. Finally, microscopic analysis of the spun bile sediments from each experimental group were similar. Numerous factors may prevent the investigator from obtaining the ideal intraoperative bile sample. Unless the operating surgeon has a particular interest in the research, he or she may be unwilling to cooperate with precholecystectomy sampling. Intraoperative gallbladder aspiration may be viewed as an unwarranted interruption of the operative procedure and introduces the potential risk of spilling bile into the sterile operative field. Complete aspiration of the in situ gallbladder may be difficult to achieve. Incomplete aspiration may not produce a representative sample as bile is often stratified in the gallbladder, and adequate mixing of bile through manipulation of the in situ gallbladder may be difficult to achieve (3, 4). With the increasing frequency of laparoscopic cholecystectomy, the problems with bile aspiration prior to cholecystectomy 1234
may be amplified. Mixing of the gallbladder contents, controlling a leaking puncture site, and dissection of a fully decompressed gallbladder become more difficult with the laparoscopic approach. Anerobic aspiration of the excised gallbladder is much easier to perform, avoids the risk of bile spillage, and makes complete aspiration of gallbladder bile more likely. The observed differences in pH, total protein, and phospholipids may be due to several factors. The consistent increase in pH in samples obtained after cholecystectomy may be explained by a decrease in the ischemic mucosa's ability to either secrete hydrogen ions or to maintain a hydrogen ion gradient (11). The decrease may also be explained by contamination of the bile with blood, which is of a more alkaline pH. The increase in biliary protein and the decrease in total phospholipids may signify enrichment of the postcholecystectomy samples with bile adjacent to the mucosal surface. Such bile is rich in the glycoprotein, mucin (12). The effects of stratification of bile in the gallbladder lumen may also play a role with the postcholecystectomy samples containing more of the denser protein and less of the lower-density phospholipids. In this study we attempted to minimize stratification effects by comparing an initial partial aspirate of gallbladder bile obtained after gentle mixing of the gallbladder contents with a completion aspirate after cholecystectomy. In conclusion, gallbladder bile sampling is an important step in research into gallstone pathogenesis. Bile samples are often obtained immediately after cholecystectomy from the ischemic gallbladder. This technique introduces changes in gallbladder bile pH, protein, and phospholipid from samples obtained prior to operative manipulation of the gallbladder. These changes are relatively small in magnitude when compared to other sampling techniques such as endoscopic sampling of bile-rich duodenal fluid (2); however, they may influence results in current biliary research. These results demonstrate the importance of maintaining a consistent sampling technique within a given study and also suggest that sampling technique be reported to facilitate the comparison of results between studies. REFERENCES 1. Strasberg SM, Harvey PRC, Hofmann AF: Bile sampling, processing, and analysis in clinical studies. Hepatology 12"176S-182S, 1990 Digestive Diseases and Sciences, Vol. 37, No. 8 (August 1992)
E F F E C T OF C H O L E C Y S T E C T O M Y ON BILE COMPOSITION 2. Janowitz P, Swobodnik W, Wechsler JG, Zoller A, Kuhn K, Ditschunheit H: Comparison of gallbladder bile and endoscopically obtained duodenal bile. Gut 31:1407-1410, 1990 3. Campbell BA, Burton AC: Stratification of bile in the gallbladder and cholelithiasis. Surg Gynecol Obstet 88:731738, 1949 4. Tera H: Stratification of human gallbladder bile in vivo. Acta Chir Scand 256(suppl):4-85, 1960 5. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254, 1976 6. Dryer RL, Tammes AR, Routh JI: The determination of phosphorus and phosphatase with N-phenyl-p-phenylenediamine. J Biol Chem 255:177-183, 1956
Digestive Diseases and Sciences, Vol. 37, No. 8 (August 1992)
7. Roschlau P, Bernt E, Gruber W: Cholesterol and esterified cholesterol. Methods Enzym Anal 4:1890-1893, 1974 8. Talalay P: Enzymatic analysis of steroid hormones. Methods Biochem Anal 8:119-143, 1960 9. Carey MC: Critical tables for calculating the cholesterol saturation of native bile. J Lipid Res 19:945-955, 1978 10. Spivak W, Yeuy W: Application of a rapid and efficienth.p.l.c. method to measure bilirubin and its conjugates from native bile and model bile systems. Biochem J 234:101-109, 1986 11. Weinman SA, Reuss L: Na§ + exchange at the apical membrane of the nectarus gallbladder: Extracellular and intracellular pH studies. J Gen Physiol 80:299-321, 1982 12. Lee SP, LaMont JT, Carey MC: Role of gallbladder mucus hypersecretion in the evolution of cholesterol gallstones. J Clin Invest 67:1712-1723, 1981
1235
Effect of Cholecystectomy on Gallbladder Bile Composition STEVEN A. AHRENDT, MD, THOMAS H. MAGNUSON, MD, HENRY A. PITT, MD, and KEITH D. LILLEMOE, MD
Human bile samples are commonly used in biliary research; however, the optimal sampling technique is not known. The current study examines whether bile obtained prior to operative manipulation of the gallbladder differs in composition from samples obtained after cholecystectomy. Gallbladder bile samples were obtained from 26 patients undergoing cholecystectomy. An initial sample was obtained prior to manipulation or devascularization of the gallbladder, and a second sample was obtained after the removal of the gallbladder from the operative field. Gallbladder bile pH and total protein were significantly increased in the postcholecystectomy samples. Bile obtained after cholecystectomy also contained significantly less phospholipid. Gallbladder bile cholesterol, total bile acids, bilirubin, ionized and total calcium, cholesterol saturation index, and total lipids were similar between techniques. These results indicate that manipulation of the gallbladder during cholecystectomy produces alterations in gallbladder bile composition. These results also emphasize the need for consistent sampling technique when obtaining samples for biliary research. KEY WORDS: cholecystectomy; bile; bile composition; bile protein; bile pH.
The analysis of gallbladder bile provides essential information in studies of the pathogenesis of gallstones. Obtaining adequate human bile samples is an important step in the experimental process. Gallbladder bile may be obtained by direct percutaneous aspiration, via endoscopic aspiration of bile-rich duodenal fluid, or from the gallbladder at laparotomy (1, 2). Of these three methods only intraoperative sampling produces representative, minimally contaminated samples without additional risk to the patient or study subject. Ideally, intraoperative bile sampling should be performed prior Manuscript received September 16, 1991; revised manuscript received February 7, 1992; accepted February 27, 1992. From the Department of Surgery, The Johns Hopkins Medical Institutions, Baltimore, Maryland. Supported in part by N.I.H. Grant R29-DK41889 from the National Institutes of Diabetes, Digestive, and Kidney Diseases (K.D.L.) and a Veterans AdministrationMerit Review (H.A.P.). Address for reprint requests: Dr. Keith D. Lillemoe, The Johns Hopkins Hospital, 600 N. Wolfe Street, Blalock 656, Baltimore, Maryland 21205.
1232
to manipulation of the gallbladder, which may introduce blood and cellular debris into the bile (1). Moreover, sampling after cholecystectomy results in the collection of bile after the gallbladder has been rendered ischemic from ligation of the cystic artery. Yet due to technical considerations of surgery, bile samples prior to manipulation may be difficult to obtain. In addition, incomplete aspiration of the in situ gallbladder may not produce a representative sample due to the stratification of bile within the gallbladder (3, 4). Despite these theoretical objections to using samples obtained after operative manipulation of the gallbladder, few data exist comparing bile obtained before with bile obtained after cholecystectomy. The present study was undertaken to determine whether gallbladder bile obtained prior to operative manipulation of the gallbladder was different in biochemical composition from samples obtained after removal of the gallbladder from the operative field. Digestive Diseases and Sciences, Vol. 37, No. 8 (August 1992)
0163-2116D2g0800-1232506.50/09 1992PlenumPublishingCorporation
EFFECT OF CHOLECYSTECTOMY ON BILE COMPOSITION TABLE 1. GALLBLADDERBILE COMPOSITIONBEFOREAND AFTEROPERATIVE MANIPULATIONOF GALLBLADDER Precholecystectomy
pH Total protein (mg/ml) Total bile acids (mM) Total bile acids (molar %) Phospholipids (mM) Phospholipids (molar %) Cholesterol (mM) Cholesterol (molar %) Cholesterol saturation index Total lipids (g/dl) Total bilirubin (~M) Unconjugated bilirubin (~M) Bilirubin monoglucuronide (I~M) Bilirubin diglucuronide (I~M) Ionized calcium (mM) Total calcium (mM)
Postcholecystectomy
6.94 --- 0.07 9.5 --- 2.4 122 --- 18 70.7 -+ 1.6 39 +- 6 21.7 --- 1.3 9.6 -+ 1.3 7.7 1.5 1.42 +- 0.36 12.7 --- 1.7 1930 --- 340 52.7 -+ 17 482 --- 95 1400 --- 250 1.90 -+ 0.23 7.58 -+ 1.0
7.14 --+ 17.4 --113 --72.3 30 19.9 --9.2 +7.7 +1.56 --11.4
-+
0.06a* 3.2b 18 1.2 4b 0.8 1.3 0.8 0.35 1.6
1600 - 290 64.5 -+ 17 391 --- 98 1140 --- 210 1.66 --+ 0.19 7.12 4- 0.9
*a, P < 0.001, and b, P < 0.01 versus precholecystectomy.
M A T E R I A L S AND M E T H O D S Bile samples were obtained from 26 patients undergoing elective, open cholecystectomy. Twenty-two patients had symptomatic gallstones (18 cholesterol and four pigment). The remaining four patients underwent cholecystectomy associated with other hepatobiliary procedures and did not have gallstones. In all patients an initial bile sample was obtained prior to placing clamps on the gallbladder or proceeding with the dissection. The gallbladder was initially manually manipulated in an attempt to mix the gallbladder contents. A 16-gauge needle was inserted into the gallbladder fundus, and 5 ml of bile were anerobically aspirated. Standard cholecystectomy was then performed. Following cholecystectomy the gallbladder was removed from the operative field, and the remaining gallbladder bile was completely aspirated. All bile samples were light-protected and immediately analyzed for ionized calcium and pH using an ion specific electrode (534 Ca2+/pH analyzer, Ciba-Corning, Medfield, Massachusetts). The remaining bile was then centrifuged for 5 min at 2000 rpm and the supernatant stored at - 2 0 ~ C. An aliquot of supernatant (75 ~1) was stored separately with ascorbic acid (1:1 v/v) in sealed light-protected vials for the later determination of bilirubin. After complete bile aspiration, the gallbladder was opened and representative gallstones selected for analysis. A spun bile sediment from both bile samples was examined under the polarizing light microscope for evidence of calcium bilirubinate granules or cholesterol crystals. All bile samples were subsequently rewarmed to room temperature and analyzed for total protein, biliary lipids, bilirubin, and total calcium. Total biliary protein was determined using the Bio-Rad assay (5). Phospholipids were quantitated using the method of Dryer et al (6). Cholesterol concentration was determined using the method of Roschlau et al (7). Total bile acids were measured using the technique of Talalay (8). The cholesterol saturation index was calculated using Carey's critical tables (9). Conjugated, unconjugated, and total billDigestive Diseases and Sciences, Vol. 37, No. 8 (August 1992)
rubin were measured by high-performance liquid chromatography as described by Spivak et al (10). Biliary total calcium was determined spectrophotometrically. Data are presented as the mean +-- SEM. Statistical analysis was performed using Student's t test (two-tailed) for paired data.
RESULTS Gallbladder bile c o m p o s i t i o n in the precholecyst e c t o m y and p o s t c h o l e c y s t e c t o m y samples are presented in Table 1. Biliary p H in the samples prior to any gallbladder manipulation was significantly less than the p H in samples obtained immediately after c h o l e c y s t e c t o m y (P < 0.001). This trend was present in 20 of the 24 samples in which the p H was measured. Biliary protein concentration was markedly increased in samples obtained after r e m o v a l of the gallbladder w h e n c o m p a r e d to samples prior to c h o l e c y s t e c t o m y (P < 0.01). Gallbladder bile obtained after operative manipulation contained significantly (P < 0.01) less phospholipid than bile obtained prior to c h o l e c y s t e c t o m y . Bile cholesterol concentrations and total bile acid concentrations were not affected b y the timing of the sample. The molar percent phospholipid, cholesterol, and bile acid and the ratio o f bile acids to phospholipids were not significantly altered b y the timing o f the bile sample. In addition, the cholesterol saturation index and the total lipid concentration were not affected b y the o p e r a t i v e handling of the gallbladder. Bilirubin monoglucuronide, bilirubin diglucuronide, unconjugated bilirubin, and total bilirubin w e r e similar b e t w e e n the p r e c h o l e c y s t e c t o m y and
1233
AHRENDTET AL postcholecystectomy samples. Operative handling and devascularization likewise did not affect ionized or total calcium concentrations. Microscopic examination of the spun bile sediment in 21 patients revealed cholesterol crystals in three patients, calcium bilirubinate granules in four patients, and both cholesterol monohydrate and calcium bilirubinate crystals in nine patients. In only one of the patients examined did the microscopic exam differ between the two samples. This patient had calcium bilirubinate granules in the sample obtained after cholecystectomy but not in the sample obtained prior to handling the gallbladder. DISCUSSION Accurate, representative bile sampling is crucial in conducting valid research in gallstone pathogenesis. The present study compares two commonly used techniques of handling intraoperative gallbladder bile samples to determine if differences in biochemical composition or microscopic analysis exist between bile obtained prior to handling the gallbladder and bile obtained after cholecystectomy. The results demonstrate that biliary cholesterol, total bile acids, and total and ionized calcium were similar between the pre- and postcholecystectomy samples. In addition, the cholesterol saturation index was not significantly affected by the sampling technique. However, biliary pH, total protein, and phospholipid concentrations did differ between the two groups. Finally, microscopic analysis of the spun bile sediments from each experimental group were similar. Numerous factors may prevent the investigator from obtaining the ideal intraoperative bile sample. Unless the operating surgeon has a particular interest in the research, he or she may be unwilling to cooperate with precholecystectomy sampling. Intraoperative gallbladder aspiration may be viewed as an unwarranted interruption of the operative procedure and introduces the potential risk of spilling bile into the sterile operative field. Complete aspiration of the in situ gallbladder may be difficult to achieve. Incomplete aspiration may not produce a representative sample as bile is often stratified in the gallbladder, and adequate mixing of bile through manipulation of the in situ gallbladder may be difficult to achieve (3, 4). With the increasing frequency of laparoscopic cholecystectomy, the problems with bile aspiration prior to cholecystectomy 1234
may be amplified. Mixing of the gallbladder contents, controlling a leaking puncture site, and dissection of a fully decompressed gallbladder become more difficult with the laparoscopic approach. Anerobic aspiration of the excised gallbladder is much easier to perform, avoids the risk of bile spillage, and makes complete aspiration of gallbladder bile more likely. The observed differences in pH, total protein, and phospholipids may be due to several factors. The consistent increase in pH in samples obtained after cholecystectomy may be explained by a decrease in the ischemic mucosa's ability to either secrete hydrogen ions or to maintain a hydrogen ion gradient (11). The decrease may also be explained by contamination of the bile with blood, which is of a more alkaline pH. The increase in biliary protein and the decrease in total phospholipids may signify enrichment of the postcholecystectomy samples with bile adjacent to the mucosal surface. Such bile is rich in the glycoprotein, mucin (12). The effects of stratification of bile in the gallbladder lumen may also play a role with the postcholecystectomy samples containing more of the denser protein and less of the lower-density phospholipids. In this study we attempted to minimize stratification effects by comparing an initial partial aspirate of gallbladder bile obtained after gentle mixing of the gallbladder contents with a completion aspirate after cholecystectomy. In conclusion, gallbladder bile sampling is an important step in research into gallstone pathogenesis. Bile samples are often obtained immediately after cholecystectomy from the ischemic gallbladder. This technique introduces changes in gallbladder bile pH, protein, and phospholipid from samples obtained prior to operative manipulation of the gallbladder. These changes are relatively small in magnitude when compared to other sampling techniques such as endoscopic sampling of bile-rich duodenal fluid (2); however, they may influence results in current biliary research. These results demonstrate the importance of maintaining a consistent sampling technique within a given study and also suggest that sampling technique be reported to facilitate the comparison of results between studies. REFERENCES 1. Strasberg SM, Harvey PRC, Hofmann AF: Bile sampling, processing, and analysis in clinical studies. Hepatology 12"176S-182S, 1990 Digestive Diseases and Sciences, Vol. 37, No. 8 (August 1992)
E F F E C T OF C H O L E C Y S T E C T O M Y ON BILE COMPOSITION 2. Janowitz P, Swobodnik W, Wechsler JG, Zoller A, Kuhn K, Ditschunheit H: Comparison of gallbladder bile and endoscopically obtained duodenal bile. Gut 31:1407-1410, 1990 3. Campbell BA, Burton AC: Stratification of bile in the gallbladder and cholelithiasis. Surg Gynecol Obstet 88:731738, 1949 4. Tera H: Stratification of human gallbladder bile in vivo. Acta Chir Scand 256(suppl):4-85, 1960 5. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254, 1976 6. Dryer RL, Tammes AR, Routh JI: The determination of phosphorus and phosphatase with N-phenyl-p-phenylenediamine. J Biol Chem 255:177-183, 1956
Digestive Diseases and Sciences, Vol. 37, No. 8 (August 1992)
7. Roschlau P, Bernt E, Gruber W: Cholesterol and esterified cholesterol. Methods Enzym Anal 4:1890-1893, 1974 8. Talalay P: Enzymatic analysis of steroid hormones. Methods Biochem Anal 8:119-143, 1960 9. Carey MC: Critical tables for calculating the cholesterol saturation of native bile. J Lipid Res 19:945-955, 1978 10. Spivak W, Yeuy W: Application of a rapid and efficienth.p.l.c. method to measure bilirubin and its conjugates from native bile and model bile systems. Biochem J 234:101-109, 1986 11. Weinman SA, Reuss L: Na§ + exchange at the apical membrane of the nectarus gallbladder: Extracellular and intracellular pH studies. J Gen Physiol 80:299-321, 1982 12. Lee SP, LaMont JT, Carey MC: Role of gallbladder mucus hypersecretion in the evolution of cholesterol gallstones. J Clin Invest 67:1712-1723, 1981
1235
