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Hypercalcemia decreases bile flow and increases biliary calcium in the prairie dog
Hypercalcemia decreases bile flow and increases biliary calcium in the prairie dog Steven A. Ahrendt, MD, Gretchen M. Ahrendt, MD, Henry A. Pitt, MD, Edward W. Moore, MD, and Keith D. Lillemoe, MD, Baltimore, Md., and Richmond, Va. Background. Biliary calcium is known to play an important role in the pathogenesis of gallstones. Calcium salts are present in all pigment gallstones and are also present in the core of most, if not all, cholesterol gallstones. Methods. The effects of acute hypercalcemia on bile flow and biliary calcium secretion were examined in 22 prairie dogs during intravenous taurocholate infusion (0, 1.0, 2.25, and 4.5 #mol/kg/min) . Results. Bile flow was linearly correlated with bile acid output in both control (y = 7.62x + 13.5, r = 0.98) and hypercalcemic (y = 7.00x + lO.d, r = 0.96) animals. At lower bile acid outputs (<3.0 #mol/kg/min), biliary ionized calcium output per increment bile acid output was significantly increased in hypercalcemic animals (0.016 versus 0.011 #tool Ca ++ ~mol taurocholate, p < 0.001). Bile ionized calcium concentrations approximated Gibbs-Donnan predicted values only at low bile flow rate. Conclusions. Hypercalcemia decreases bile flozo and increases biliary ionized calcium concentration in the prairie dog. These effects favor the precipitation of calcium salts in bile. (SURGERY 1995; 117:435-42.) From the Department of Surgery, The Johns Hopkins Medical Institutions, Baltimore, Md., and the Department of Medicine, The Medical College of Virginia, Richmond, Va.
CALCIUM PLAYS A CRITICAL role in the formation of
most, if not all, gallstones. Precipitation of the calcium salts of bilirubinate, carbonate, phosphate, and palmitate is a necessary step in the pathogenesis of pigment gallstones] Moreover, calcium salts have been found at the core of most cholesterol gallstones and may play a role in their formation. 2' 3 T h e addition of calcium to dilute model bile accelerates cholesterol-containing phospholipid vesicle aggregation and cholesterol monohydrate crystal nucleation. 4 Evidence also suggests that oral calcium supplementation increases biliary calcium in the prairie dog model and prolonged administration leads to pigment gallstone formation. 5' 6 These findings suggest that an increase in the secretion of calcium into bile may promote gallstone formation. Despite the significance of the calcium ion in gallstone pathogenesis, the complex interrelationships between biliary and serum free ionized calcium, bile salt concenSupported in part by National Institutes of Health grant R-29-DK 41889. Presented in part at the 91st Annual Meeting of the American Gastroenterologieal Association, San Antonio, Texas, 1990. Accepted for publication Sept. 2, 1994. Reprint requests: Keith D. Lillemoe, MD, The Johns Hopkins Hospital, Department of Surgery, 600 N. Wolfe St., Blalock 603, Baltimore, MD 21287. Copyright 9 1995 by Mosby-Year Book, Inc. 0039-6060/95/$3.00 + 0 11/56/60468
tration, and bile flow remain incompletely defined. Free calcium ions enter canalicular bile passively across paracellular channels in response to bile salt secretion.V, s Theoretically, biliary ionized calcium concentration is a function of serum ionized calcium and biliary bile salt concentration as determined by Gibbs-Donnan forces. 9 In these experiments we sought to examine the effect of hypercalcemia on bile flow and biliary calcium secretion in the prairie dog, a commonly used model of gallstone pathogenesis.
MATERIAL AND METHODS Twenty-two adult male prairie dogs (Cynomys ludovicianus) trapped in the wild and obtained from Otto Martin Locke, New Braunfels, Texas, were used in this study. All the animals received a standard, nonlithogenie diet (Purina Rodent Chow; Purina Mills, St. Louis, Mo.) for at least 2 weeks before the short-term experiments. Experimental design. After an overnight fast allowing water ad libitum, animals were anesthetized with 100 m g / k g intramuscular ketamine, and a laparotomy was performed through a midline incision. T h e cystic duct was ligated and a cholecystectomy was performed. T h e distal common bile duct was ligated and cannulated proximally with a polyethylene-50 catheter. Hepatic bile was collected in tared vials on ice. Both femoral veins were cannulated with polyethylene-50 tubing for SURGERY
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Table. Effect of hypercalcemia on ionized calcium and bile acid secretion, bile flow, and bile calcium binding
Group Normocalcemia Hypercalcemia Normocalcemia Hypercalcemia Normocalcemia Hypercalcemia
Taurocholate rate (ttmol/kg/min) n 0 0 2.25 2.25 4.5 4.5
4 4 4 4 2 2
Serum {Ca ++] (mmol/L) 1.30 _+ 0.03 1.89 _+ 0.04* 1.21 _+ 0.04 1.90 • 0.10" 1.60 _+ 0.02 2.59 • 0.21t
Bile [Ca++] (mmol/L)
[Ca ++] Bile acid output Bile flow output (tzmol/kg/rnin) (#l/kg/min) (~trnol/kg/min)
1.21 _+ 0.02 0.019 • 0.001 2.22 _+ 0.11~ 0.024 • 0.002 1.12 _+ 0.04 0.038 _+ 0.001 2.00 _+ 0.07qk 0.054 • 0.002~ 1.50 • 0.04w 0.075 + 0.002 1.94 + 0.18t 0.066 + 0.003
15.5 + 0.4 10.6 + 0.8t 33.8 _+ 1.0 27.1 _+ 0.7~ 50.6 _+ 2.3 35.0 • 1.6:~
0.26 _+ 0.03 0.22 _+ 0.02 2.32 _+ 0.07 2.16 _+ 0.07 5.05 • 0.26 3.89 _+ 0.17~:
Bile [Ca++]/ [Ca T] 0.70 _+ 0.01 0.59 • 0.02:[: 0.72 _+ 0.01 0.63 • 0.011: 0.75 • 0.02 0.63 • 0.01:[:
Values expressed as mean + SEM. *p < 0.05, t P < 0.01, ~:p < 0.001 vs normocaleemic controls at equal bile acid infusion rates. w < 0.05 vs normocalcemic controls at 0 ,umol/kg/min taurocholate.
the infusion of fluid and pharmacologic agents. All animals received a total infused volume of normal saline solution at 0.4 ml/min at all times to maintain hemodynamic stability throughout the length of these experiments. Animals were maintained at 37 ~ C throughout the experiment with an infrared lamp. After insertion of the common duct cannula, all animals underwent bile collection for 2 hours during which depletion of the bile acid pool occurred. After completion of this bile acid depletion period, either normal saline solution or sodium taurocholate (98% pure; Sigma Chemical Company, St. Louis, Mo.) was infused at a constant rate of 1.0, 2.25, or 4.5 #mol/kg/min for 3 hours. The prairie dog liver secretes only taurine conjugates of the primary bile acids, cholate and chenodeoxycholate, with taurocholate comprising nearly 90% of the bile salt pool] ~ In 10 animals acute hypercalcemia was induced after the 2-hour depletion period with a 0.11 mmol/kg intravenous loading dose of calcium gluconate during a 5-minute period followed by an intravenous infusion at 0.005 mmol/kg/min for 3 hours. The remaining 12 normocalcemic control animals received an equivalent volume of normal saline solution. Within 30 minutes of the start of the taurocholate infusion, bile flow had stabilized. Hepatic bile samples were then collected at either 15- or 30-minute intervals as needed to ensure sufficient sample size for analysis. Bile flow was determined gray• Serum samples for total and ionized calcium were obtained hourly through the femoral venous cannula. Bile analysis. Whole blood and hepatic bile were immediately analyzed for ionized calcium by using ion specific electrodes (Ciba-Corning 634 Ca ++ pH analyzer; Ciba Corning Diagnostics Corp., Medfield, Mass.). The remaining bile and blood were centrifuged for 5 minutes at 2000 rpm, and the supernatant was stored at - 2 0 ~ C. After rewarming to room temperature, bile and serum were analyzed for total calcium spectrophotometrically, and total bile acids were measured by using the method of Talalay] 1
Data analysis. Results are expressed as mean _+ SEM. Comparisons between groups were performed with analysis of variance. Correlations were made with linear regression analysis by the method of least squares. Slopes and y-intercepts of regression lines were compared with analysis of variance. Significance was accepted at the p < 0.05 level.
RESULTS Serum and hepatic bile ionized calcium concentrations, biliary ionized calcium output, bile flow, biliary total bile acid output, and percent of total calcium present as free calcium ions are shown in the Table. Data are presented for both normocalcemic and hypercalcemic animals at varying taurocholate infusion rates. Calcium gluconate infusion significantly increased both serum and bile ionized calcium concentrations as expected. Bile ionized calcium concentration was significantly (p < 0.05) increased in the normocalcemic animals receiving the taurocholate infusion at 4.5 #mol/ kg/min when compared with animals receiving no taurocholate. A similar increase in bile ionized calcium concentration was not observed in the normocalcemic animals receiving the bile acid infusion at 2.25 ~mol/ kg/min or in the hyperealcemic animals. Ionized calcium output, bile flow, and bile acid output all increased as the bile acid infusion rate increased. Bile ionized calcium output was significantly increased in hypercalcemic animals receiving taurocholate at 2.25 #mol/kg/ min when compared with normocalcemic animals at the same infusion rate; however, at the higher bile acid infusion (4.5 #mol/kg/min) this effect with hypercalcemia no longer existed. Serum and bile total calcium concentrations and outputs paralleled those of ionized calcium (data not shown). Bile flow was significantly decreased in hypercalcemic animals at all taurocholate infusion rates. Bile acid output was decreased in hypercalcemic animals receiving taurocholate at 4.5 #mol/kg/min when compared with normocalcemic controls. This decrease did not
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reach significance at the lower bile acid infusion rates. Finally, the percent of total biliary calcium present as free calcium ions was significantly decreased by the calcium infusion. Bile flow versus bile acid output. The relationship between bile flow and bile acid output is illustrated in Fig. 1. Bile flow was linearly related to bile acid output for both normocalcemic (y = 7.62x + 13.5, r = 0.98, p < 0.001) and hypercalcemic (y = 7.00x + 10.42, r = 0.96, p < 0.001) animals. The slope of the lines is a measure of the osmotic potency of a given micellar bile acid and has been designated the apparent choleretic activity. 12 Hypercaleemia did not appear to have any effect on the apparent choleretic activity of taurocholate. The fraction of bile flow induced by the osmotic activity of premicellar bile acids can be calculated by extrapolating these regression lines to the y-intercept. Hypercalcemia significantly (p < 0.001) decreased this fraction of bile flow. Ionized calcium output versus bile acid output. The effect of hypercalcemia on biliary ionized calcium output is shown in Fig. 2. A significant correlation existed between ionized calcium output and bile acid output both in the normocalcemic (y = 0.011x + 0.015, r = 0.96, p < 0.001) and in the hypercalcemic animals (y = 0.016x + 0.019, r = 0.86, p < 0.001) at bile acid outputs <3.0 #mol/kg/min. A baseline bile acid output in the fasted prairie dog was determined from the first 30-minute bile sample obtained from each animal during the bile acid depletion period. This baseline bile acid output was 0.81 _+ 0.15 #mol/kg/min. None of the baseline values was greater than 3.0 ~mol/kg/min. Hypercalcemia significantly increased (p < 0.001) ion-
ized calcium output per increment bile acid output at bile acid outputs <3.0 #mol/kg/min. The entry of the calcium ion into bile is passive and at equilibrium is determined largely by Gibbs-Donnan forces created mainly by the impermeant bile acids present in bile. The Gibbs-Donnan ratio, g, relates bile to serum activity ratios for calcium and has been determined for taurocholate by Mooreg: g = [Ca++]V:B/[Ca++]'/2s = 1 + 0.0025 [BA-] (1) where BA is bile acid. Thus [Ca++]s = [Ca++]s + 0.005 [Ca++]s[BA-] + 6.25 X 10 -6 [Ca++]s [BA-] 2 (2) In the prairie dog hepatic bile acid concentrations vary from 40 to 65 mmol/L between fasted and nonfasted animals. 13 At these concentrations the [BA-] 2 term makes up less than 2% of biliary calcium, and the relationship between bile calcium concentration and serum calcium and biliary bile acid concentration is nearly linear: [Ca++]B = [Ca++]s + 0.005 [Ca++]s [BA-] (3) The predicted increase in ionized calcium output with increases in bile acid output was observed in hypercalcemic animals (Fig. 2). Observed versus predicted bile calcium. With this relationship (equation 2) the Gibbs-Donnan predicted values for bile calcium concentration were calculated from the serum calcium and bile acid concentrations. The relationship between observed and Gibbs-Donnan predicted values for hepatic bile ionized calcium concentrations is shown in Fig. 3 for samples collected at
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bile flow rates less than 25 #1/kg/min. Observed bile calcium concentrations closely paralleled (y = 0.99x - 0.087, r = 0.82, n -- 19) predicted values at these lower flow rates. At bile flow rates exceeding 25 #1/kg/ min no correlation existed between observed and predicted ionized calcium concentrations. The effect of bile flow on Gibbs-Donnan predicted values for bile calcium concentration is shown in Fig. 4. At lower bile flow rates observed calcium concentrations approximated predicted values, and most points fell next
to the unity line. At higher bile flow rates (>25 # l / k g / min) observed calcium concentrations were less than Gibbs-Donnan predicted values. Bile calcium versus s e r u m calcium. T h e effect of serum ionized calcium on biliary ionized calcium is illustrated in Fig. 5. At equilibrium, biliary calcium is theoretically dependent on serum calcium and the concentration of impermeable anions in bile (equation 3), necessitating examination of this relationship at a single bile acid concentration. Values plotted are all simul-
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taneous serum and bile calcium determinations in bile acid-depleted (bile acid output <0.2 # m o l / k g / m i n ) , normocalcemic, and hypercalcemic animals. Bile ionized calcium was linearly related to serum ionized calcium (y = 0.89x + 0.08, r = 0.86, p < 0.001) in bile acid-depleted animals (Fig. 5) in which G i b b s - D o n n a n effects are minimal. Bile total calcium was also linearly
related to serum total calcium (y = 1 . 1 7 x - 0 . 2 5 , r = 0.95, p < 0.001). Bile flow v e r s u s s e r u m c a l c i u m . T h e effect of serum calcium on bile flow is graphically represented in Fig. 6 for bile acid-depleted animals (bile acid output <0.2 # m o l / k g / m i n ) . A significant inverse relationship was observed between bile flow and serum ionized calcium
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Fig. 7. Effect of hypercalcemia and bile acid concentration on percent of total calcium present in ionized form, [Ca++]/[CaT], in bile. Percent of unbound calcium is decreased in hypercalcemia (n -- 48) when compared with normocalcemic animals (n = 98). Percent of unbound calcium was not affected by changes in bile acid concentration. (y = 2 8 - 9.6x, r = 0.68, p < 0.01) (Fig. 6) and between bile flow and serum total calcium (y = 19 - 2.2x, r = 0.50, p < 0.05) (not shown). C a l c i u m b i n d i n g . T h e ratio of free ionized calcium ions to total calcium present in bile is depicted in Fig. 7 for a wide range of bile acid concentrations. H y p e r calcemia decreased the percent of calcium present in ionized form when compared with normocalcemic controls. Varying bile acid concentration throughout the micellar range (10 to 120 m m o l / L ) did not appreciably alter this binding for either the normocalcemic or hy-
percalcemic animals. Below the critical micellar concentration of taurocholate, the percent of total calcium present in ionized form appeared to significantly increase in the normocalcemic animals, although few samples were collected in this range. D I S C U S S I O N
T h e present study was designed to further delineate the role of calcium ions in biliary secretion. H y p e r c a l cemia significantly increased bile ionized calcium concentration and output in the prairie dog. Bile flow was
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significantly decreased in hypercalcemic animals. Calcium infusion also tended to decrease bile acid output in taurocholate-infused animals. Moreover, the percent of calcium bound to buffers in bile also increased with the increased entry of calcium into bile. At low bile flow rates, observed values for bile ionized calcium concentration were similar to Gibbs-Donnan predicted values (equation 2). As bile flow increases, calcium entry into bile is limited, and calcium outputs fall below predicted Gibbs-Donnan values. Hypercalcemia significantly reduced bile flow at all bile acid infusion rates without consistently altering bile acid output. A decrease in bile flow with hypercalcemia has previously been shown in the cat by Layer et al., 14 although at significantly greater serum calcium concentrations. Limlongwongse et al., 15 however, were unable to demonstrate any significant change in bile flow in hypercalcemic rats. In addition, in the in vitro perfused rat liver, hepatic uptake and secretion of taurocholate are independent of extracellular calcium) 6,17 In the present study hypercalcemia decreased bile flow despite having no effect on the apparent choleretic activity of micellar bile acids. Several investigators have demonstrated that bile flow varies with bile acid excretion in a curvilinear fashion)2,18,19 Below the critical micellar concentration (CMC), the slope of the line relating bile flow to bile acid output is steeper than above the CMC, reflecting the markedly greater osmotic activity of monomeric bile acids. 2~We were unable to achieve premicellar bile acid concentrations to demonstrate this curvilinear decrease in this study. Despite a 5-hour period of bile acid depletion in the animals receiving no taurocholate, the mean bile acid concentration in the normocalcemic animals was 16 mmol/L, significantly greater than the C M C of taurocholate (about 5 to 6 mmol/L). Recently, Moore and Sanyal zl have demonstrated that high affinity binding of calcium to premicellar bile acids (BA) occurs as dimeric Ca (BA)2 complexes. Enhanced dimer formation in hypercalcemia may decrease the osmotic activity of premicellar bile acids and thus decrease bile flow. Calcium-dependent changes in the C M C may also lead to alterations in bile flow. Moore and Sanyal have demonstrated that the C M C for glycocholate in the presence of calcium is less than in calcium-free solutions, indicating that the calcium ion promotes micelle formation. The C M C of other bile acids is also strongly dependent on total ionic and ambient calcium concentration.22, 23 A decrease in the C M C as the calcium ion concentration in bile increases would also lower the concentration of the more osmotically active bile acid monomers, thus leading to a decrease in bile flow. Further studies examining the role of free calcium ions on the C M C of various bile acids are clearly indicated. In addition to its effect on bile flow, hypercalcemia
Ahrendt et al.
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also significantly increased bile ionized calcium output. Several investigators have demonstrated a linear relationship between calcium output and bile acid output at a single serum calcium concentration and have concluded that calcium enters bile passively) 2,24,25 Studies examining the entry of radiolabeled calcium into bile have also suggested that calcium enters bile rapidly across paracellular channels. 26 Biliary calcium concentrations should thus be determined by Gibbs-Donnan forces created by impermeant anions present in bile. However, the Gibbs-Donnan equation has not been verified in vivo by varying both serum ionized calcium and biliary bile acid concentrations, the determinants of bile ionized calcium concentration (equation 2). These data showing the close correlation between observed and predicted values for ionized calcium output at only lower bile flow rates emphasize the role of additional factors in determining biliary ionized calcium concentrations. Rege et alJ 2 have stressed the importance of bile flow and residence time in determining whether GibbsDonnan predicted values for ionized calcium concentration are attained in the dog. A strong dependence of biliary calcium concentration on bile flow has also been exhibited in human beings. 2v In bile acid-depleted animals bile flow rates are low, and biliary ionized calcium approximated serum calcium concentration, indicating adequate time for passive equilibration to occur (Fig. 4). We did not observe a significant increase in hepatic bile ionized calcium concentration when compared with serum ionized calcium as bile acid concentration increased until the taurocholate infusion rate was 4.5 #mol/kg/min. Other investigators have also demonstrated that bile calcium concentration remains relatively constant over a wide range of bile acid eoneentrations both in human beings and in the dog) 2, 24 This observation may indicate that insufficient time for complete equilibration of calcium between serum and bile exists even at relatively low bile flow rates. In the present study values for bile ionized calcium concentration fell below Gibbs-Donnan predicted values at bile flow rates normally observed in nonfasted prairie dogs (25 u l / k g / m i n ) J 3 Significant protection against calcium precipitation is provided by numerous calcium buffers in bile. The binding of calcium to bile salts to form soluble complexes in bile is important in limiting increases in ionized calcium concentration. 28"3~ Conjugated bilirubin, bicarbonate, and proteins including mucin have subsequently been shown to participate in the binding of calcium in bile.31, 32 At all bile acid concentrations examined, hypercalcemia increased the amount of calcium bound to calcium buffers in bile. Moore 1 has previously demonstrated that bile acids, the major buffer of calcium in bile, form a high capacity binding system for calcium
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ions. By binding relatively m o r e calcium in hypercalcemia, the rise in the concentration of free calcium ions in bile is attenuated, thus partially decreasing the likelihood of calcium precipitation. T h e s e e x p e r i m e n t s suggest that h y p e r c a l c e m i a m a y increase bile lithogenesis by increasing biliary ionized calcium o u t p u t w h i l e decreasing bile flow. T h e s e effects favor the precipitation of calcium salts in bile. E n h a n c e d binding of calcium to various buffers present in bile in h y p e r c a l c e m i a partially protects against increases in ionized calcium concentration. C a l c i u m entry into bile closely conforms to G i b b s - D o n n a n predicted values only at low bile flow rates. As bile acid concentration and bile flow increase, calcium entry into bile does not reach e q u i l i b r i u m and bile calcium concentrations do not reach G i b b s - D o n n a n predicted values. We acknowledge the technical expertise of Karen FoxTalbot. REFERENCES
1. Moore EW. The role of calcium in the pathogenesis of gallstones: Ca ++ electrode studies of model bile salt solutions and other biologic systems. Hepatology 1984;4:228S-43S. 2. Been JM, Bills PM, Lewis D. Microstructure of gallstones. Gastroenterology 1979;76:548-55. 3. Kaufman HS, Lillemoe KD, Magnuson TH, Frasca PF, Pitt HA. Backscattered electron imaging and windowless energy dispersive x-ray microanalysis: a new technique for gallstone analysis. Scanning Microse 1990;4:853-62. 4. Kibe A, Dudley MA, Halpern Z, Lynn MP, Breuer AC, Holzbach RT. Factors affecting cholesterol monohydrate crystal nucleation time in model systems of supersaturated bile. J Lipid Res 1985;26:1102-11. 5. Lillemoe KD, Magnuson TH, Peoples GE, Li L, Pitt HA. Oral calcium supplementation increases biliary calcium [Abstract]. Gastroenterology 1988;94:A563. 6. Magnuson TH, Lillemoe KD, Peoples GE, Pitt HA. Oral calcium promotes pigment gallstone formation. J Surg Res 1989; 46:286-91. 7. Graf J. Canalicular bile salt-independent bile formation: concepts and clues from electrolyte transport in rat liver. Am J Physiol 1983;244:G233-46. 8. Moore EW, Rasmusen H, Shiffman M. Pathogenesis of calcium-containing gallstones: all biliary calcium in the guinea pig is accounted for by passive entry from plasma [Abstract]. Gastroenterology 1986;90:1748. 9. Moore EW. The regulation of ionized calcium [Ca++], in bile: taurocholate induces powerful Gibbs-Donnan effects in vitro [Abstract]. Gastroenterology 1988;94:A572. 10. Cohen BI, Mosbach EH, McSherry CK, et al. Gallstone prevention in prairie dogs: comparison of chow vs. semisynthetic diets. Hepatology 1986;6:874-80. 11. Talalay P. Enzymic analysis of steroid hormones. In: Methods of biochemical analysis. New York: Interscience Publishers Inc, 1960:8:119-43. 12. Rege RV, Dawes LG, Moore EW. Biliary calcium secretion in the dog occurs primarily by passive convection and diffusion
Surgery April 7995 and is linked to bile flow. J Lab Clin Med 1990;115:593602. 13. Magnuson TH, Ahrendt SA, Lillemoe KD, et al. Short-term fasting increases biliary calcium and bilirubin. J Surg Res 1991;50:529-524. 14. Layer P, Hotz J, Sinewe S, Goebell H. Bile secretion in acute and chronic hypercalcemia in the cat. Dig Dis Sci 1986;31:18892. 15. Limlomwongse L, Deachapunya C, Krishnamra N. Biliary calcium and bile acid secretion in intact and TPTX rats with varying plasma calcium concentration. Dig Dis Sci 1988;33:68591. 16. Anwer MS, Clayton LM. Role of extracellular Ca ++ in hepatic bile formation and taurocholate transport. Am J Physiol 1985; 249:G711-8. 17. Reichen J, Berr F, Le M, Warren GH. Characterization of calcium deprivation-induced cholestasis in the perfused rat liver. Am J Physiol 1985;249:G48-57. 18. Baker AL, Wood RAB, Moosa AR, Boyer JL. Sodium taurocholate modifies the bile acid-independent fraction of canalicular bile flow in the rhesus monkey. J Clin Invest 1979;64:31220. 19. Blitzer BL, Boyer JL. Cellular mechanisms of bile formation. Gastroenterology 1982;82:346-57. 20. Moore EW. Bile salt osmotic activity [Abstract]. Hepatology 1986;6:1157. 21. Moore EW, Sanyal AJ. Ca++-binding to bile acids: I. Affinity and stoichiometry. Hepatology 1989;10:731. 22. Van der Meet R, Vonk RJ, Kuipers F. Cholestasis and the interaction of sulfated glyco- and taurolithocholate with calcium. Am J Physiol 1988;254:G644-9. 23. Roda A, Hofmann AF, Mysels KJ. The influence of bile salt structure on self-association in aqueous solutions. J Biol Chem 1983;258:6362-70. 24. Knyrim K, Vakil N, Pfab R, Classen M. The effects of intraduodenal bile acid administration on biliary secretion of ionizedcalcium and carbonate in man. Hepatology 1989;10:134-42. 25. Cummings SA, Hofmann AF. Physiologic determinants of biliary calcium secretion in the dog. Gastroenterology 1984;87:66473. 26. Jaeschke H, Benzick AE. Paracellular and transcellular movement of calcium from blood into bile [Abstract]. Hepatology 1989;10:729. 27. Knyrim K, Vakil N. The effects of biliary obstruction on ductal ionized calcium concentrations in man [Abstract]. Hepatology 1988;8:1362. 28. Moore EW, Celic L, Ostrow JD. Interactions between ionized calcium and sodium taurocholate: bile salts are important buffers for prevention of calcium-containing gallstones. Gastroenterology 1982;83:1079-89. 29. Rajagopalan N, Lindenbaum S. The binding of Ca ++ to taurine- and glycine-conjugated bile salt micelles. Biochim Biophys Acta 1982;711:66-74. 30. Williamson BWA, Percy-Robb IW. Contribution of biliary lipids to calcium binding in bile. Gastroenterology 1980;78:696702. 31. Shimizu S, Rege RV, Webster CC, Ostrow JD, Celic L, Moore EW. High-affinity binding of ionized calcium (Ca ++) by conjugated bilirubin [Abstract]. Hepatology 1987;7:1110. 32. Dawes LG, Rege RV, Moore EW. Bile salts, bilirubin, and carbonate: the major biliary binders of calcium [Abstract]. Hepatology 1988;8:1258.
CALCIUM PLAYS A CRITICAL role in the formation of
most, if not all, gallstones. Precipitation of the calcium salts of bilirubinate, carbonate, phosphate, and palmitate is a necessary step in the pathogenesis of pigment gallstones] Moreover, calcium salts have been found at the core of most cholesterol gallstones and may play a role in their formation. 2' 3 T h e addition of calcium to dilute model bile accelerates cholesterol-containing phospholipid vesicle aggregation and cholesterol monohydrate crystal nucleation. 4 Evidence also suggests that oral calcium supplementation increases biliary calcium in the prairie dog model and prolonged administration leads to pigment gallstone formation. 5' 6 These findings suggest that an increase in the secretion of calcium into bile may promote gallstone formation. Despite the significance of the calcium ion in gallstone pathogenesis, the complex interrelationships between biliary and serum free ionized calcium, bile salt concenSupported in part by National Institutes of Health grant R-29-DK 41889. Presented in part at the 91st Annual Meeting of the American Gastroenterologieal Association, San Antonio, Texas, 1990. Accepted for publication Sept. 2, 1994. Reprint requests: Keith D. Lillemoe, MD, The Johns Hopkins Hospital, Department of Surgery, 600 N. Wolfe St., Blalock 603, Baltimore, MD 21287. Copyright 9 1995 by Mosby-Year Book, Inc. 0039-6060/95/$3.00 + 0 11/56/60468
tration, and bile flow remain incompletely defined. Free calcium ions enter canalicular bile passively across paracellular channels in response to bile salt secretion.V, s Theoretically, biliary ionized calcium concentration is a function of serum ionized calcium and biliary bile salt concentration as determined by Gibbs-Donnan forces. 9 In these experiments we sought to examine the effect of hypercalcemia on bile flow and biliary calcium secretion in the prairie dog, a commonly used model of gallstone pathogenesis.
MATERIAL AND METHODS Twenty-two adult male prairie dogs (Cynomys ludovicianus) trapped in the wild and obtained from Otto Martin Locke, New Braunfels, Texas, were used in this study. All the animals received a standard, nonlithogenie diet (Purina Rodent Chow; Purina Mills, St. Louis, Mo.) for at least 2 weeks before the short-term experiments. Experimental design. After an overnight fast allowing water ad libitum, animals were anesthetized with 100 m g / k g intramuscular ketamine, and a laparotomy was performed through a midline incision. T h e cystic duct was ligated and a cholecystectomy was performed. T h e distal common bile duct was ligated and cannulated proximally with a polyethylene-50 catheter. Hepatic bile was collected in tared vials on ice. Both femoral veins were cannulated with polyethylene-50 tubing for SURGERY
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Table. Effect of hypercalcemia on ionized calcium and bile acid secretion, bile flow, and bile calcium binding
Group Normocalcemia Hypercalcemia Normocalcemia Hypercalcemia Normocalcemia Hypercalcemia
Taurocholate rate (ttmol/kg/min) n 0 0 2.25 2.25 4.5 4.5
4 4 4 4 2 2
Serum {Ca ++] (mmol/L) 1.30 _+ 0.03 1.89 _+ 0.04* 1.21 _+ 0.04 1.90 • 0.10" 1.60 _+ 0.02 2.59 • 0.21t
Bile [Ca++] (mmol/L)
[Ca ++] Bile acid output Bile flow output (tzmol/kg/rnin) (#l/kg/min) (~trnol/kg/min)
1.21 _+ 0.02 0.019 • 0.001 2.22 _+ 0.11~ 0.024 • 0.002 1.12 _+ 0.04 0.038 _+ 0.001 2.00 _+ 0.07qk 0.054 • 0.002~ 1.50 • 0.04w 0.075 + 0.002 1.94 + 0.18t 0.066 + 0.003
15.5 + 0.4 10.6 + 0.8t 33.8 _+ 1.0 27.1 _+ 0.7~ 50.6 _+ 2.3 35.0 • 1.6:~
0.26 _+ 0.03 0.22 _+ 0.02 2.32 _+ 0.07 2.16 _+ 0.07 5.05 • 0.26 3.89 _+ 0.17~:
Bile [Ca++]/ [Ca T] 0.70 _+ 0.01 0.59 • 0.02:[: 0.72 _+ 0.01 0.63 • 0.011: 0.75 • 0.02 0.63 • 0.01:[:
Values expressed as mean + SEM. *p < 0.05, t P < 0.01, ~:p < 0.001 vs normocaleemic controls at equal bile acid infusion rates. w < 0.05 vs normocalcemic controls at 0 ,umol/kg/min taurocholate.
the infusion of fluid and pharmacologic agents. All animals received a total infused volume of normal saline solution at 0.4 ml/min at all times to maintain hemodynamic stability throughout the length of these experiments. Animals were maintained at 37 ~ C throughout the experiment with an infrared lamp. After insertion of the common duct cannula, all animals underwent bile collection for 2 hours during which depletion of the bile acid pool occurred. After completion of this bile acid depletion period, either normal saline solution or sodium taurocholate (98% pure; Sigma Chemical Company, St. Louis, Mo.) was infused at a constant rate of 1.0, 2.25, or 4.5 #mol/kg/min for 3 hours. The prairie dog liver secretes only taurine conjugates of the primary bile acids, cholate and chenodeoxycholate, with taurocholate comprising nearly 90% of the bile salt pool] ~ In 10 animals acute hypercalcemia was induced after the 2-hour depletion period with a 0.11 mmol/kg intravenous loading dose of calcium gluconate during a 5-minute period followed by an intravenous infusion at 0.005 mmol/kg/min for 3 hours. The remaining 12 normocalcemic control animals received an equivalent volume of normal saline solution. Within 30 minutes of the start of the taurocholate infusion, bile flow had stabilized. Hepatic bile samples were then collected at either 15- or 30-minute intervals as needed to ensure sufficient sample size for analysis. Bile flow was determined gray• Serum samples for total and ionized calcium were obtained hourly through the femoral venous cannula. Bile analysis. Whole blood and hepatic bile were immediately analyzed for ionized calcium by using ion specific electrodes (Ciba-Corning 634 Ca ++ pH analyzer; Ciba Corning Diagnostics Corp., Medfield, Mass.). The remaining bile and blood were centrifuged for 5 minutes at 2000 rpm, and the supernatant was stored at - 2 0 ~ C. After rewarming to room temperature, bile and serum were analyzed for total calcium spectrophotometrically, and total bile acids were measured by using the method of Talalay] 1
Data analysis. Results are expressed as mean _+ SEM. Comparisons between groups were performed with analysis of variance. Correlations were made with linear regression analysis by the method of least squares. Slopes and y-intercepts of regression lines were compared with analysis of variance. Significance was accepted at the p < 0.05 level.
RESULTS Serum and hepatic bile ionized calcium concentrations, biliary ionized calcium output, bile flow, biliary total bile acid output, and percent of total calcium present as free calcium ions are shown in the Table. Data are presented for both normocalcemic and hypercalcemic animals at varying taurocholate infusion rates. Calcium gluconate infusion significantly increased both serum and bile ionized calcium concentrations as expected. Bile ionized calcium concentration was significantly (p < 0.05) increased in the normocalcemic animals receiving the taurocholate infusion at 4.5 #mol/ kg/min when compared with animals receiving no taurocholate. A similar increase in bile ionized calcium concentration was not observed in the normocalcemic animals receiving the bile acid infusion at 2.25 ~mol/ kg/min or in the hyperealcemic animals. Ionized calcium output, bile flow, and bile acid output all increased as the bile acid infusion rate increased. Bile ionized calcium output was significantly increased in hypercalcemic animals receiving taurocholate at 2.25 #mol/kg/ min when compared with normocalcemic animals at the same infusion rate; however, at the higher bile acid infusion (4.5 #mol/kg/min) this effect with hypercalcemia no longer existed. Serum and bile total calcium concentrations and outputs paralleled those of ionized calcium (data not shown). Bile flow was significantly decreased in hypercalcemic animals at all taurocholate infusion rates. Bile acid output was decreased in hypercalcemic animals receiving taurocholate at 4.5 #mol/kg/min when compared with normocalcemic controls. This decrease did not
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reach significance at the lower bile acid infusion rates. Finally, the percent of total biliary calcium present as free calcium ions was significantly decreased by the calcium infusion. Bile flow versus bile acid output. The relationship between bile flow and bile acid output is illustrated in Fig. 1. Bile flow was linearly related to bile acid output for both normocalcemic (y = 7.62x + 13.5, r = 0.98, p < 0.001) and hypercalcemic (y = 7.00x + 10.42, r = 0.96, p < 0.001) animals. The slope of the lines is a measure of the osmotic potency of a given micellar bile acid and has been designated the apparent choleretic activity. 12 Hypercaleemia did not appear to have any effect on the apparent choleretic activity of taurocholate. The fraction of bile flow induced by the osmotic activity of premicellar bile acids can be calculated by extrapolating these regression lines to the y-intercept. Hypercalcemia significantly (p < 0.001) decreased this fraction of bile flow. Ionized calcium output versus bile acid output. The effect of hypercalcemia on biliary ionized calcium output is shown in Fig. 2. A significant correlation existed between ionized calcium output and bile acid output both in the normocalcemic (y = 0.011x + 0.015, r = 0.96, p < 0.001) and in the hypercalcemic animals (y = 0.016x + 0.019, r = 0.86, p < 0.001) at bile acid outputs <3.0 #mol/kg/min. A baseline bile acid output in the fasted prairie dog was determined from the first 30-minute bile sample obtained from each animal during the bile acid depletion period. This baseline bile acid output was 0.81 _+ 0.15 #mol/kg/min. None of the baseline values was greater than 3.0 ~mol/kg/min. Hypercalcemia significantly increased (p < 0.001) ion-
ized calcium output per increment bile acid output at bile acid outputs <3.0 #mol/kg/min. The entry of the calcium ion into bile is passive and at equilibrium is determined largely by Gibbs-Donnan forces created mainly by the impermeant bile acids present in bile. The Gibbs-Donnan ratio, g, relates bile to serum activity ratios for calcium and has been determined for taurocholate by Mooreg: g = [Ca++]V:B/[Ca++]'/2s = 1 + 0.0025 [BA-] (1) where BA is bile acid. Thus [Ca++]s = [Ca++]s + 0.005 [Ca++]s[BA-] + 6.25 X 10 -6 [Ca++]s [BA-] 2 (2) In the prairie dog hepatic bile acid concentrations vary from 40 to 65 mmol/L between fasted and nonfasted animals. 13 At these concentrations the [BA-] 2 term makes up less than 2% of biliary calcium, and the relationship between bile calcium concentration and serum calcium and biliary bile acid concentration is nearly linear: [Ca++]B = [Ca++]s + 0.005 [Ca++]s [BA-] (3) The predicted increase in ionized calcium output with increases in bile acid output was observed in hypercalcemic animals (Fig. 2). Observed versus predicted bile calcium. With this relationship (equation 2) the Gibbs-Donnan predicted values for bile calcium concentration were calculated from the serum calcium and bile acid concentrations. The relationship between observed and Gibbs-Donnan predicted values for hepatic bile ionized calcium concentrations is shown in Fig. 3 for samples collected at
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bile flow rates less than 25 #1/kg/min. Observed bile calcium concentrations closely paralleled (y = 0.99x - 0.087, r = 0.82, n -- 19) predicted values at these lower flow rates. At bile flow rates exceeding 25 #1/kg/ min no correlation existed between observed and predicted ionized calcium concentrations. The effect of bile flow on Gibbs-Donnan predicted values for bile calcium concentration is shown in Fig. 4. At lower bile flow rates observed calcium concentrations approximated predicted values, and most points fell next
to the unity line. At higher bile flow rates (>25 # l / k g / min) observed calcium concentrations were less than Gibbs-Donnan predicted values. Bile calcium versus s e r u m calcium. T h e effect of serum ionized calcium on biliary ionized calcium is illustrated in Fig. 5. At equilibrium, biliary calcium is theoretically dependent on serum calcium and the concentration of impermeable anions in bile (equation 3), necessitating examination of this relationship at a single bile acid concentration. Values plotted are all simul-
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taneous serum and bile calcium determinations in bile acid-depleted (bile acid output <0.2 # m o l / k g / m i n ) , normocalcemic, and hypercalcemic animals. Bile ionized calcium was linearly related to serum ionized calcium (y = 0.89x + 0.08, r = 0.86, p < 0.001) in bile acid-depleted animals (Fig. 5) in which G i b b s - D o n n a n effects are minimal. Bile total calcium was also linearly
related to serum total calcium (y = 1 . 1 7 x - 0 . 2 5 , r = 0.95, p < 0.001). Bile flow v e r s u s s e r u m c a l c i u m . T h e effect of serum calcium on bile flow is graphically represented in Fig. 6 for bile acid-depleted animals (bile acid output <0.2 # m o l / k g / m i n ) . A significant inverse relationship was observed between bile flow and serum ionized calcium
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percalcemic animals. Below the critical micellar concentration of taurocholate, the percent of total calcium present in ionized form appeared to significantly increase in the normocalcemic animals, although few samples were collected in this range. D I S C U S S I O N
T h e present study was designed to further delineate the role of calcium ions in biliary secretion. H y p e r c a l cemia significantly increased bile ionized calcium concentration and output in the prairie dog. Bile flow was
Surgery Volume 117, Number d
significantly decreased in hypercalcemic animals. Calcium infusion also tended to decrease bile acid output in taurocholate-infused animals. Moreover, the percent of calcium bound to buffers in bile also increased with the increased entry of calcium into bile. At low bile flow rates, observed values for bile ionized calcium concentration were similar to Gibbs-Donnan predicted values (equation 2). As bile flow increases, calcium entry into bile is limited, and calcium outputs fall below predicted Gibbs-Donnan values. Hypercalcemia significantly reduced bile flow at all bile acid infusion rates without consistently altering bile acid output. A decrease in bile flow with hypercalcemia has previously been shown in the cat by Layer et al., 14 although at significantly greater serum calcium concentrations. Limlongwongse et al., 15 however, were unable to demonstrate any significant change in bile flow in hypercalcemic rats. In addition, in the in vitro perfused rat liver, hepatic uptake and secretion of taurocholate are independent of extracellular calcium) 6,17 In the present study hypercalcemia decreased bile flow despite having no effect on the apparent choleretic activity of micellar bile acids. Several investigators have demonstrated that bile flow varies with bile acid excretion in a curvilinear fashion)2,18,19 Below the critical micellar concentration (CMC), the slope of the line relating bile flow to bile acid output is steeper than above the CMC, reflecting the markedly greater osmotic activity of monomeric bile acids. 2~We were unable to achieve premicellar bile acid concentrations to demonstrate this curvilinear decrease in this study. Despite a 5-hour period of bile acid depletion in the animals receiving no taurocholate, the mean bile acid concentration in the normocalcemic animals was 16 mmol/L, significantly greater than the C M C of taurocholate (about 5 to 6 mmol/L). Recently, Moore and Sanyal zl have demonstrated that high affinity binding of calcium to premicellar bile acids (BA) occurs as dimeric Ca (BA)2 complexes. Enhanced dimer formation in hypercalcemia may decrease the osmotic activity of premicellar bile acids and thus decrease bile flow. Calcium-dependent changes in the C M C may also lead to alterations in bile flow. Moore and Sanyal have demonstrated that the C M C for glycocholate in the presence of calcium is less than in calcium-free solutions, indicating that the calcium ion promotes micelle formation. The C M C of other bile acids is also strongly dependent on total ionic and ambient calcium concentration.22, 23 A decrease in the C M C as the calcium ion concentration in bile increases would also lower the concentration of the more osmotically active bile acid monomers, thus leading to a decrease in bile flow. Further studies examining the role of free calcium ions on the C M C of various bile acids are clearly indicated. In addition to its effect on bile flow, hypercalcemia
Ahrendt et al.
441
also significantly increased bile ionized calcium output. Several investigators have demonstrated a linear relationship between calcium output and bile acid output at a single serum calcium concentration and have concluded that calcium enters bile passively) 2,24,25 Studies examining the entry of radiolabeled calcium into bile have also suggested that calcium enters bile rapidly across paracellular channels. 26 Biliary calcium concentrations should thus be determined by Gibbs-Donnan forces created by impermeant anions present in bile. However, the Gibbs-Donnan equation has not been verified in vivo by varying both serum ionized calcium and biliary bile acid concentrations, the determinants of bile ionized calcium concentration (equation 2). These data showing the close correlation between observed and predicted values for ionized calcium output at only lower bile flow rates emphasize the role of additional factors in determining biliary ionized calcium concentrations. Rege et alJ 2 have stressed the importance of bile flow and residence time in determining whether GibbsDonnan predicted values for ionized calcium concentration are attained in the dog. A strong dependence of biliary calcium concentration on bile flow has also been exhibited in human beings. 2v In bile acid-depleted animals bile flow rates are low, and biliary ionized calcium approximated serum calcium concentration, indicating adequate time for passive equilibration to occur (Fig. 4). We did not observe a significant increase in hepatic bile ionized calcium concentration when compared with serum ionized calcium as bile acid concentration increased until the taurocholate infusion rate was 4.5 #mol/kg/min. Other investigators have also demonstrated that bile calcium concentration remains relatively constant over a wide range of bile acid eoneentrations both in human beings and in the dog) 2, 24 This observation may indicate that insufficient time for complete equilibration of calcium between serum and bile exists even at relatively low bile flow rates. In the present study values for bile ionized calcium concentration fell below Gibbs-Donnan predicted values at bile flow rates normally observed in nonfasted prairie dogs (25 u l / k g / m i n ) J 3 Significant protection against calcium precipitation is provided by numerous calcium buffers in bile. The binding of calcium to bile salts to form soluble complexes in bile is important in limiting increases in ionized calcium concentration. 28"3~ Conjugated bilirubin, bicarbonate, and proteins including mucin have subsequently been shown to participate in the binding of calcium in bile.31, 32 At all bile acid concentrations examined, hypercalcemia increased the amount of calcium bound to calcium buffers in bile. Moore 1 has previously demonstrated that bile acids, the major buffer of calcium in bile, form a high capacity binding system for calcium
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ions. By binding relatively m o r e calcium in hypercalcemia, the rise in the concentration of free calcium ions in bile is attenuated, thus partially decreasing the likelihood of calcium precipitation. T h e s e e x p e r i m e n t s suggest that h y p e r c a l c e m i a m a y increase bile lithogenesis by increasing biliary ionized calcium o u t p u t w h i l e decreasing bile flow. T h e s e effects favor the precipitation of calcium salts in bile. E n h a n c e d binding of calcium to various buffers present in bile in h y p e r c a l c e m i a partially protects against increases in ionized calcium concentration. C a l c i u m entry into bile closely conforms to G i b b s - D o n n a n predicted values only at low bile flow rates. As bile acid concentration and bile flow increase, calcium entry into bile does not reach e q u i l i b r i u m and bile calcium concentrations do not reach G i b b s - D o n n a n predicted values. We acknowledge the technical expertise of Karen FoxTalbot. REFERENCES
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