Editorial APF/CBP, An Anionic Polypeptide in Bile and Gallstones That May Regulate Calcium Salt and Cholesterol Precipitation from Bile The article in this issue of HE;PATOLOGY by Kestell et al. (1) is a welcome confirmation of the presence in bile and gallstones of a small anionic calcium-binding biliprotein -APFiCBP (anionic polypeptideicalcium binding protein) - origmally discovered by Lafont and her co-workers in Marseille (2, 3 ) as a result of their interest in hepatic lipoprotein transport and, later, in our laboratory in Chicago (4, 5 ) as a result of our hypothesis that gallstones are a biomineralization system (4, 6). This editorial will review the properties and possible roles of this protein in the regulation of the precipitation of calcium salts from bile (and thus in formation of all types of gallstones). I hope it will rectify the preoccupation with cholesterol precipitation that has dominated gallstone research for the past two decades. The possible role of APF/CBP in hepatic cholesterol and lipid transport will also be mentioned. Normal human bile is always thermodynamically supersaturated with the calcium salts of bilirubinate Ca(HB), (7,8) and often supersaturated with carbonate (9, 10) and cholesterol (9, 11).These calcium salts and others are present in all cholesterol gallstones, including their cores (6, 12),and in all pigment gallstones (12, 13). Thus one must ask, as with cholesterol (141, what inhibits precipitation of these calcium salts from normal bile and what promotes their precipitation to form gallstones in pathological bile (12).Normal bile contains a preponderance of lunetic factors that potently inhibit cholesterol precipitation ( 15). whereas pathological bile contains a preponderance of factors that potently promote (16) cholesterol Precipitation. The major kinetic factors for cholesterol are an array of bile glycoproteins, including mucin (17),that promote (17-20)or inhibit (15, 21, 22) nucleation, or growth, of cholesterol crystals. Though calcium is not required for the lunetic effects of bile proteins on cholesterol precipitation t23), variations in [Ca” 1 modulate these effects ( 2 4 ) ; however, it is not known whether t.hese proteins interact with Ca2 . Although bile contains factors that inhibit precipitation of calcium salts (25-28~,only one bileigallstone protein with such an effect has been described. Our +
This work was supported by a Medical Investigator Award from thr I! S Department of Veterans Affairs and hy extramural research grant 2 - K O I - D K 32130 from the National Institutes of Health Address reprint requests to. J . Donald Ostrow. M.D.. Medical Investigator. Research Service (151).D.V.A. Lakeside iMedic-al Center. 410 East Ontario Street, Chicago, IL 6061 1 3 1/1/42668
laboratory initially isolated this calcium-binding protein from “pure” and “mixed” cholesterol gallstones (4) and hepatic T-tube bile (29) and, subsequently, from black pigment gallstones (5).CBP is almost certainly identical 1 30, 3 1 ) to the anionic polypeptide (APF)discovered earlier by Lafont and her co-workers as the major apoprotein of the bile pigment-lipoprotein complex in pathological gallbladder bile ( 2 , 3 )and, subsequently, in cholesterol and pigment gallstones (32).APFiCBP, the third most abundant protein in bile (Domingo N, et al., Manuscript submitted, 19921, has been shown to be the o d y protein other than mucin that is present in all gallstones (4, 5, 31, 32) and in all normal and pathological biles (29-31; Domingo N, et al., Manuscript submitted, 1992). It is one of the few proteins that precipitates on addition of CaC1, to bile at pH 8.0, and it potently inhibits the precipitation of CaCO, from supersaturated solutions in uztro ( 4 , 2 9 , 3 1 )APF/CBP . also promotes the permeability and fusion of lecithincholesterol vesicles and the nucleation of cholesterol from model biles (33).It is the first bileigallstone protein shown to affect precipitation of both calcium salts and cholesterol and thus likely has a key role in the formation of pigment and cholesterol gallstones. Properties of APFICBP. APF and CBP from gallstones and from bile have many common properties and, as noted above, are likely identical (2-5, 30, 31). Both yield bands with apparent molecular weights of 6.5 and 12 kD on SDS-PAGE in gels containing 1 5 9 to 16.5% acrylamide. Neither stains with Coomassie blue, but each stains orange-brown with silver and blue with Stains-All. Both are highly anionic proteins containing approximately 24% acidic and 8% basic amino acids, a high proportion of serine (some phosphorylated), threonine and neutral amino acids and low contents of methionine and cysteine. Amino acid analyses also show variable high glycine contents, likely related to contaminating glycoconjugated bile salts, as suggested by the presence also of taurine from taurine-conjugated bile salts. The amphipathic properties of both proteins engender avid self-aggregation and strong binding of phospholipids, bile pigments and bile salts. The pigments are in part covalently bound, gwing the protein a yellow-to-brown color that is much darker in CBP isolated from gallstones than in APF from bile. This binding of bile pigments may account for the puzzling finding of soluble bilirubin conjugates in pigment gallstones (34).Variation in types and amounts of bound bilirubins account for the presence of three differently colored bands on
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isoelectric focusing (PI = 4.5,4.7and 4.9) (2, 30). Interaction with added phospholipids alters the mobility of the protein on zone electrophoresis (2).Most convincing is that APF and CBP exhibit identical, unique immunostaining on Western blotting and react identically with a panel of one polyclonal antibody and seven monoclonal antibodies (MAbs) prepared against APF and one polyclonal antibody and four MAbs against apolipoprotein A-I (APoA-I)(31). Although APF/CBP was initially thought to be a glycoprotein (4), purer preparations of APF/CBP are unaffected by endoglycosidases F or H and contain no sugars (asdetermined on gas-capillary chromatography after acid hydrolysis) and consequently do not bind to concanavalin A (conA) or other lectins (Kannicht C, et al., Manuscript submitted, 1992). Nonetheless, when untreated bile is applied to a conA column, a proportin of APF/CBP is retained and removed subsequently by the specific eluant, a-methyl mannoside (Lafont H, Personal communication, 1992). This behavior may be due to binding of APF/CBP to the glycoproteins and amphiphilic compounds that adsorb directly to the conA. Other investigators have usually not detected APF/ CBP in bile, conA-adsorbed fractions of bile or biliary vesicles. This is due mainly to the low molecular weight (7 kD) of APF/CBP, which is lost, therefore, by ultrafiltration and dialysis through devices with pores that allow passage of proteins of 10 kD or less, by running off the ends of gels prepared with less than 12%acrylamide, by elution from the gel during the customary prolonged fixation with poorly polar solvents and by staining gels with Coomassie blue only and not with silver. APF/CBP is detected in all biles, whether or not gallstones are present, with attention to these precautions. The study protocol used by Kestell et al. (1) parallels methods used initially by Shimizu et al. (4,5)of isolation of CBP after demineralization of cholesterol gallstones and after precipitation from bile by addition of 0.5 m o m CaC1, (29-31). There are, however, two important differences in the procedures of Kestell et al.: the first is the apparent omission of protease inhibitors during the EDTA extraction, which are considered essential in the isolation of proteins from biomineralization systems (35). The second difference is the omission of the Sephadex G-25 gel filtration of the EDTA extract, which removes most of the lipids and other small molecules, including bile pigments, bile salts, peptides and amino acids (4,5,31),as well as calcium and EDTA. The second difference is especially important. Isolation of CPB by PAGE in a 12% acrylamide gel cannot yield pure material because the 6.5-kD peptide runs at the gel front and may contain other molecules of molecular weights less than 12 kD that also run at the front. The presence of these impurities likely accounts for major differences between their amino acid analyses and more recent preparations from other laboratories (30, 31) in which 15% to 16.5% acrylamide gels were used to purify APF/CBP and remove smaller contaminants not separated on the 12%gels, including bile pigments, bile salts, oligopeptides and amino acids. Because lipids were not removed, it is probable that Kestell et al. did not isolate APF/CBP protein but rather
HEPATOLOGY
something akin to the bile pigment-lipoprotein complex ( 2 , 3 )and biliary vesicles (36,37).The particle size of 60 nm detected on quasielastic light scattering and the minute proportion ( < 4%) of protein indicate that the isolated material is likely a lipoprotein particle (l), though the researchers inexplicably failed to analyze for lipids. Unfortunately, Kestell et al. therefore cannot attribute the observed properties of their isolated material (self-aggregation,pigment binding, calcium binding and inhibition of CaCO, precipitation) to the protein per se; rather, these might be properties of the presumed lipid components, of the bound bile salts or bile pigments or of the whole complex. For example, glycoconjugated, dihydroxy-bile salts, even at premicellar concentrations, inhibit the crystallization of calcium carbonates (38) and phosphates (39,401. Kestell et al. (1) confirm the prior reports of the presence of APF/CBP in gallbladder and hepatic bile and cholesterol and pigment gallstones, its precipitation by calcium and its binding of lipids. The major new findings are the first direct demonstrations of calcium binding by the “protein” and its strong tendency to self-aggregate, especially in the presence of calcium. As noted by the authors, however, this self-aggregation and precipitation of the lipoprotein particles precluded determination of the affinity constants for binding of calcium. Their dialysis studies have two other limitations: (a) the continued slow decline in 45Ca in the bath and the irregularity of the counts in the contents of the dialysis sac suggest that equilibrium was not attained, an event that takes many hours when precipitates are present or forming in the system (41); and (b) the failure to measure APF/CBP in the bath makes the claim that the bath was protein free inappropriate. Dialysis with a 10-kD cutoff membrane would likely allow some of the 6.5-kD APF/CBP monomer to enter the dialysate. How Might APFICBP Regulate Calcium Salt Precipitation, and Why Is I t Present in Gallstones?We believe that APF/CBP is the small regulatory polypeptide of the biomineralization system in bile (4,6,42).APF/CBP has characteristics typical of peptides that regulate calcium deposition in bones and teeth (35, 42, 43) and in renal (44) and pancreatic (45) calculi: size less than 20 kD, amphipathic and anionic character and phosphorylated serines. We propose that APF/CBP binds to the hydrophobic, unglycosylated “intermediate piece” of the large structural protein, mucin (46),and then binds Ca2 ions to its own anionic groups, which are properly spaced to act as a template for growth of the calcium salt crystals (6). In analogy to a building, mucins are the girders, APF/CBP is the mortar and the calcium salt crystals are the bricks. As the crystal grows on the matrix, more APF/CBP (by way of its anionic domains) may bind to the growth face of the calcium salt crystal. Depending on conditions, the exposed hydrophobic domain of APF/CBP could then (a) arrest further accretion of calcium salts to the growing crystal; (b) bind cholesterol, resulting in a cholesterol stone; or (c) bind more mucin, triggering a new cycle of mineralization in a new layer of the calculus. APF/CBP, like other small molecules, might diffuse into the interstices of the already formed stone (47). +
l’ltOl’ER‘l’lk~S OF .4PF CBP I N UILE AND GALLSTONES
Vol 16. No. 6. 1992
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Using MAbs for ELISA, it was determined that con- specifically with cy-methyl mannoside appears to reside centrations of APF/CBP in normal bile average 1.0 mainly in the 5-kD rather than in the 130-kD protein mg/ml (gallbladder) and 0.4 mg:’ml (hepatic);concentra- (52). Because the 5-kD protein reacts with MAbs tions relative to bile salts average 17% higher in bile prepared against APF, this protein is probably APF/CBP from patients with cholesterol gallstone5 and 29% or a large fragment thereof (Lafont H, Personal comhigher in those with pigment gallstones (Doming0 N , et munication). Clearly APF/CBP can affect precipitation al., Manuscript submitted, 1992 ) . These relatively small from bile of cholesterol and of calcium salts. Future Directions. It remains to characterize fully the increases in APF/CBP concentrations in the bile of patients with gallstone suggest that an intrinsic qualitative molecular structure of this protein, determine whether change in the protein might be found in patients with this structure is altered qualitatively in the bile of gallstone disease, akin to abnormalities in urinary neph- patients with gallstones and elucidate the mechanisms by which APF/CBP affects calcium and cholesterol rocalcin in patients with urinary tract stones (44). Role of APFICBP in Lipid Transport and Cholesterol precipitation, quality of biliary vesicles and hepatocytic Precipitution. MAbs against the N-terminal and transport of cholesterol. This includes determination in C-terminal portions (but not against the intervening model systems of whether the bound lipids, bile salts, polypeptide) of ApoA-I cross-react with APFCBP ( 31 !. bile pigments and calcium are essential to or modulate Because APF/CBP contains three amino acids and six the actions of APF/CBP on cholsterol and calcium epitopes that are not found in ApoA-I, the former cannot precipitation. Because the APF/CBP extracted from be a fragment of the latter. These two proteins are gallstones may be partly degraded during the long probably different products of a common supergene, sojourn in the gallbladder or during the extraction generated by alternative splicing of messenger RNA of the minerals from the protein, further studies transcribed by the gene. Six MAbs prepared against should use preferentially the protein isolated freshly APF, half of which also cross-react with Apo A-I, react from bile and compare APF/CBP from normal and also with epitopes on the surface of HDL3 t 3 1 ~ ,sug- abnormal biles. Peptide maps (fingerprints) and amino gesting that APF is partially exposed on the surface of acid sequences of APF/CBP preparations are needed but this plasma lipoprotein particle. Specific binding sites for have thus far been unsuccessful because of a blocked APF are present on the basolateral plasma membranes N-terminal amino acid. Ultimately, the identity and of rat hepatocytes (481, further suggesting a role for APF structure of APF/CBP and possible alterations in pain lipoprotein uptake. Indeed, hepatocytic uptake, intra- tients with gallstones will be established by sequencing cellular distribution and metabolism of cholesterol are the gene(s1. modified by addition of APF to infused liposomes containing cholesterol (49). J. DONALDOSTROW,M.D. Immunoelectron microscopy has localized APF,’CBP D.V.A. Lakeside Medical Center to the endoplasmic reticulum of rat hepatocytes but not Chicago, Illinois 60611 bile duct or gallbladder epithelia; APFKBP is synthesized by isolated rat hepatocytes (50).L k e the biliary REFERENCES lipid particles with which it is associated, hepatic secretion of APF/CBP in rats is regulated by the rate of Kestell MF. Sekijima J , Lee SP, Park HZ, Long M, Kaler EW. A secretion and hydrophobicity of secreted bile salts ( 511. calcium binding protein in bile and gallstones. HEPATOLOW 1992;16:1315-132i . The dissociation between biliary secretion of APF and Nalbone G. Lafont H. Vime JL. Domineo N. Lairon D. Chabert C. lipids when dehydrocholate is infused (51~ 1 . however, is Lechene P: et al. The apoprotein fraction of the bile lipoprotein not in agreement with the concept that APFiCBP itself complex: isolation, partial characterization and phospholipid regulates biliary lipid secretion. binding properties. Biociumie 1985:61:1029-1041. Martigne M, Domingo N. Lafont H, Nalbone G, Hauton JC. Because APF/CBP is the major apolipoprotein of the Purification of the human anionic polypeptide fraction of the bile pigment-lipoprotein complex ( 2 , 3 , . we were not apo-bile lipoprotein complex by zonal ultracentrifugation. Lipids surprised to find that APFCBP inserts itself into 1985;20:884-889. cholesterol-lecithin vesicles, causing their permeabilShimizu S, Sabsay B. Veis A, Ostrow JD. Rege RV, Dawes LG. ization, aggregation and fusion and promoting growth Isolation of an acidic protein from cholesterol gallstones which inhibits the precipitation of calcium carbonate in vitro. J Clin of cholesterol crystals in supersaturated bile (33).These Invest 1989;84:1990-1996. effects are augmented by addition of 10 mmol/L calcium Oludo M. Shimizu S, Ostrow JD. Nakayama F. Isolation of a chloride, possibly because the hydrophobic portion of calcium-regulatory protein from black pigment gallstones: simiAPF/CBP inserts itself into the electrically neutral larity with a protein from cholesterol gallstones. HEPKroLOGY 1992;15:1079-1085. lecithin-cholesterol vesicles of bile, with its anionic Ostrow JD. Unconjugated bilirubin and cholesterol gallstone groups at the vesicular surface available to react 1990;12(suppl 1 1219S-226S. formation. HEPATOLOGY bivalently with Ca2 + ( 12 1. These preliminary studies. Ostrow JD,Celic L. Apparent solubility products (K”sp) of the however, must be confirmed in experiments with calcium salts of unconjugated bilirubin (B) indicate supersatulower, more physiological concentrations of Ca2 ration of human gallbladder bile with Ca(HB),, but not CaB 1 Abstractl. HEPATOLOGY 1987;7:1111. ( 5 2 mmol/L), pretreatment of the protein with Chelex Vadali M, Ostrow JD. Mukerjee P, Celic L. Aqueous solubility to remove all Ca2 from control samples and additional products of calcium bilirubinate salts, determined by solvent controls containing added calcium but no protein. partition from chloroform into buffer aqueous solutions of CaCI,. Interestingly, the pronucleating activity for cholesterol In: Proceedings of the XI1 International Bile Acid Meeting. Basel: 1992. of the fraction of bile that is retained by conA and eluted +
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9. Shiffman ML, Sugerman HJ, Moore EW. Human mucosal gall- 32. Martigne M, Domingo N, Lechene de la Porte P, Lafont H, Hauton JC. Identification and localization of apoprotein fraction of the bile bladder function: effect of concentration and acidification of bile on lipoprotein complex in human gallstones. Scand J Gastroenterol cholesterol and calcium solubility. Gastroenterology 1990;99: 1988;23:731-737. 1452-1459. 10. Knyrim K, Vakil N. The effects of synthetic human secretin on 33. Domingo N, Groen B, Lechene P, de Bruijn M, Ostrow JD, Lafont H. Anionic polypeptide fraction (APF) avidly interacts with calcium carbonate solubility in human bile. Gastroenterology cholesteroMecithin vesicles and promotes crystal growth in model 1990;99:1445-1451. bile [Abstract]. Gastroenterology 1992;102:A802. 11. Carey MC, Small DM. The physical chemistry of cholesterol solubility in bile: relationship to gallstone formation and disso- 34. Trotman BW, Rajagopalan C, Bernstein SE. Monoconjugated bilirubin is a major component of hemolysis-induced gallstones in lution in man. J Clin Invest 1978;61:998-1026. 1988;8:919-924. 12. Moore EW. Biliary calcium and gallstone formation. HEPATOLOGY mice. HEPATOLOGY 35. Veis A. Bones and teeth. In: Piez KA, Reddi H, eds. Extracellular 1 9 9 0 ; 1 2 ( ~ ~ p1):206S-218S. pl matrix biochemistry. New York: Elseviermorth Holland Pub13. Crowther RS,Soloway RD. Pigment gallstone pathogenesis: from lishers, 1984:329-374. man to molecules. Semin Liver Dis 1990;10:171-180. 14. Holzbach RT. Recent progress in understanding cholesterol 36. Somjen GJ, Gilat T. A non-micellar mode of cholesterol transport in bile. FEBS Lett 1983;156:265-268. crystal nucleation as a precursor to human gallstone formation. 37. Ulloa N, Garrido J , Nervi F. Ultracentrifugal isolation of vesicular HEPATOLOGY 1986;6:1403-1406. carriers of biliray cholesterol in native human and rat bile. 15. Holzbach RT, Kibe A, Thiel E, Howell JH,Marsh M, Hermann RE. HEPATOLOGY 1987;7:235-244. Biliary proteins: unique inhibitors of cholesterol crystal nucle38. Marteau C. Portugal H, Pauli AM, Gerolami A. Effect of ation in human gallbladder bile. J Clin Invest 1984;73:35-45. glycoursodeoxycholate on precipitation of calcium carbonate. 16. Burnstein JM, Ilson RG, Petrunka CN, Strasberg SM. Evidence HEPATOLOGY 1985;5:1209-1212. for a potent nucleation factor in gallbladder bile of patients with 39. Van der Meer R, DeVries HT. Differential binding of glycine- and cholesterol gallstones. Gastroenterology 1983;85:801-807. taurine-conjugated bile acids to insoluble calcium phosphate. 17. Levy PF, Smith BF, Lamont JT. Human gallbladder mucin Biochem J 1985;229:265-268. accelerates in uitro nucleation of cholesterol in artificial bile. 40. Qui SM, Wen G, Hirakawa N, Soloway RD, Hong NK, Crowther Gastroenterology 1984;87:270-275. RS. Glycochenodeoxycholic acid inhibits calcium phosphate pre18. Groen AK, Noordam C, Drapers JAG, Egbers P, Jansen PLM, cipitation in vitro by preventing the transformation of amorphous Tytgat GNJ. Isolation of a potent cholesterol nucleationcalcium phosphate to calcium hydroxyapatite. J Clin Invest promoting activity from human gallbladder bile: role in the 1991;88:1265-1271. pathogenesis of gallstone disease. HEPATOLOGY 1990;11:525-533. 1984;4(suppl): 19. Harvey PRC, Upadhya GA, Strasberg SM. Immunoglobulins as 41. Nancollas GH. Crystallization in bile. HEPATOLOGY 169s-172s. nucleating proteins in the gallbladder bile of patients with 42. Veis A, Sabsay B. Bone and tooth formation: insights into cholesterol gallstones. J Biol Chem 1991;266:13996-14003. mineralization strategies. In: Westbroek P, DeJong EW, eds. 20. Groen AK. Nonmucous glycoproteins as pronucleating agents. Biomineralization and biological metal accumulation: biological HEPATOLOGY 1990;12(suppl 1):189S-l94S. and geological. Dordrecht, The Netherlands: D. Riedel Co., 21. Busch N, Holzbach RT. Crystal growth-inhibiting proteins in bile. 1982~273-284. HEPATOLOGY 1990;12(suppl 1):195S-l99S. 22. Schwarzendrube J , Nuutinen H, Abei N, Chandler K, Williams C, 43. Sabsay B, Stetler-Stevenson WG, Lechner JH, Veis A. Domain structure and sequence distribution in dentin phosphophoryn. Holzbach RT. Amino acid sequencing and immunological localBiochem J 1991;276:699-707. ization of a unique 120 kD dimeric crystal growth-inhibiting biliary glycoprotein [Abstract]. Gastroenterology 1992;102:A882. 44. Nakagawa Y, Ahmed M, Hall SL, DeGanello S, Coe FL. Isolation from human calcium oxalate renal stones of a glycoprotein 23. Gallinger S, Harvey PRC, Petrunka CN, Strasberg SM. Effect of inhibitor of calcium oxalate crystal growth: evidence that nephbinding of ionized calcium on the in vitro nucleation of cholesterol rocalcin from patients with calcium oxalate nephrolithiasis is and calcium bilirubinate in human gallbladder bile. Gut 1986;27: deficient in y-carboxyglutamic acid. J Clin Invest 1987;79:17821382-1386. 1787. 24. Shermak MA, Lipsett PA, Kaufman HS, Fox-Talbot MK, Pitt HA, Lillemoe KD. Calcium interacts with biliary proteins to induce 45. DeCaro A, Multigner L, Lafont H, Lombard0 D, Sarles H. The molecular characteristics of a human pancreatic acidic phosphocholesterol crystal nucleation [Abstract]. Gastroenterology 1992; protein that inhibits calcium carbonate crystal growth. Biochem J 102:A941. 1984;222:669-677. 25. Sutor DJ, Percival JM. Presence or absence of inhibitors of crystal growth in bile. 1. Effect of bile on the formation of calcium 46. Smith BF, Lamont JT. Hydrophobic binding properties of bovine gallbladder mucin. J Biol Chem 1984;259:12170-12177. phosphate, a constituent of gallstones. Gut 1976;17:506-510. 26. Sutor DJ, Percival JM. The effect of bile on the crystallization of 47. Sanabria JR,Upadhya GA, Harvey PRC, Strasberg SM. Diffusion of substances into cholesterol gallstones [Abstract]. Gastroentercalcium carbonate. Clin Chim Ada 1978;89:479-484. ology 1992;102:A878. 27. Sciaretta A, Ligabue A, Garuti G, Pieromaldi S, Verri I, Giacobazzi G, Malaguti P. Inhibitory activity of gallbladder bile on calcium 48. Martigne M, Meli B, Mahlberg F, Domingo N, Chanussot F, Lafont H, Hauton JC. Detection and characterization of anionic polypepcarbonate crystallization in vitro: a comparison between normal tidic fraction binding sites in rat liver plasma membranes and subjects and gallstone patients. Scand J Gastroenterol 1984;19: cultured hepatocytes. Biochim Biophys Acta 1989;979:341-346. 626-630. 28. Dawes LG, Moore EW, Rege RV, Shimizu S, Ostrow JD. Canine 49. Martigne M, Domingo N, Chanussot F, Nalbone G, Lafont H, Hauton J . Effects of bile anionic polypeptide fraction on the fate bile contains anticrystallization factors that inhibit precipitation of cholesterol carried by liposomes in the rat. Proc Soc Exp Biol of calcium carbonate. HEPATOLOGY 1991;14:701-706. Med 1988;187:229-234. 29. Shimizu S, Sabsay B, Veis A, Ostrow JD, Dawes LG, Rege RV. A 12-kDa acidic protein from cholesterol gallstones inhibits precip- 50. Domingo N, Botta D, Martigne-Cros M, Lechene de la Porte P, Pak-Leung P, Hauton J , Lafont H. Evidence for the synthesis and itation of calcium carbonate and is present in human bile secretion of APF - a bile lipid associated protein - by isolated rat [Abstract]. HEPATOLOGY 1988;8:1257. hepatocytes. Biochim Biophys Acta 1990;1044:243-248. 30. Lafont H, Ostrow JD, Shimizu S,Domingo N, Lechene P, Hauton JC, Rege RV, et al. Are anionic polypeptide fraction (APF) from 51. Chanussot F, Domingo N, Tuchweber B, Lafont H, Yousef IB. Influence of dehydrocholic and cholic acids infusions on the biliary bile and calcium-binding protein (CBP) from gallstones identical secretion of anionic polypeptide fraction, the major apoprotein of [Abstract]? Gastroenterology 1991;100:A764. the biliary lipoprotein complex. Scand J Gastroenterol 1992;27: 31. Domingo N, Grosclaude J , Bekaert ED, Mege D, Chapman MJ, 238-242. Shimizu S, AyraultJanier M, et al. Epitope mapping of the human biliary amphipathic anionic polypeptide (APF): similarity 52. DeBruijn MAC, Mok K, Doming0 N, Lafont H, Tytgat GNJ, Groen AK. Further characterization of the cholesterol crystallization with a calcium-binding protein (CBP) isolated from gallstones and promoting lipoprotein in biliary Con-A-binding protein [Abbile, and immunological cross-reactivity with apolipoprotein A-I. stract]. Gastroenterology 1992;102:A310. J Lipid Res 1992;33:1419-1430.
This work was supported by a Medical Investigator Award from thr I! S Department of Veterans Affairs and hy extramural research grant 2 - K O I - D K 32130 from the National Institutes of Health Address reprint requests to. J . Donald Ostrow. M.D.. Medical Investigator. Research Service (151).D.V.A. Lakeside iMedic-al Center. 410 East Ontario Street, Chicago, IL 6061 1 3 1/1/42668
laboratory initially isolated this calcium-binding protein from “pure” and “mixed” cholesterol gallstones (4) and hepatic T-tube bile (29) and, subsequently, from black pigment gallstones (5).CBP is almost certainly identical 1 30, 3 1 ) to the anionic polypeptide (APF)discovered earlier by Lafont and her co-workers as the major apoprotein of the bile pigment-lipoprotein complex in pathological gallbladder bile ( 2 , 3 )and, subsequently, in cholesterol and pigment gallstones (32).APFiCBP, the third most abundant protein in bile (Domingo N, et al., Manuscript submitted, 19921, has been shown to be the o d y protein other than mucin that is present in all gallstones (4, 5, 31, 32) and in all normal and pathological biles (29-31; Domingo N, et al., Manuscript submitted, 1992). It is one of the few proteins that precipitates on addition of CaC1, to bile at pH 8.0, and it potently inhibits the precipitation of CaCO, from supersaturated solutions in uztro ( 4 , 2 9 , 3 1 )APF/CBP . also promotes the permeability and fusion of lecithincholesterol vesicles and the nucleation of cholesterol from model biles (33).It is the first bileigallstone protein shown to affect precipitation of both calcium salts and cholesterol and thus likely has a key role in the formation of pigment and cholesterol gallstones. Properties of APFICBP. APF and CBP from gallstones and from bile have many common properties and, as noted above, are likely identical (2-5, 30, 31). Both yield bands with apparent molecular weights of 6.5 and 12 kD on SDS-PAGE in gels containing 1 5 9 to 16.5% acrylamide. Neither stains with Coomassie blue, but each stains orange-brown with silver and blue with Stains-All. Both are highly anionic proteins containing approximately 24% acidic and 8% basic amino acids, a high proportion of serine (some phosphorylated), threonine and neutral amino acids and low contents of methionine and cysteine. Amino acid analyses also show variable high glycine contents, likely related to contaminating glycoconjugated bile salts, as suggested by the presence also of taurine from taurine-conjugated bile salts. The amphipathic properties of both proteins engender avid self-aggregation and strong binding of phospholipids, bile pigments and bile salts. The pigments are in part covalently bound, gwing the protein a yellow-to-brown color that is much darker in CBP isolated from gallstones than in APF from bile. This binding of bile pigments may account for the puzzling finding of soluble bilirubin conjugates in pigment gallstones (34).Variation in types and amounts of bound bilirubins account for the presence of three differently colored bands on
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isoelectric focusing (PI = 4.5,4.7and 4.9) (2, 30). Interaction with added phospholipids alters the mobility of the protein on zone electrophoresis (2).Most convincing is that APF and CBP exhibit identical, unique immunostaining on Western blotting and react identically with a panel of one polyclonal antibody and seven monoclonal antibodies (MAbs) prepared against APF and one polyclonal antibody and four MAbs against apolipoprotein A-I (APoA-I)(31). Although APF/CBP was initially thought to be a glycoprotein (4), purer preparations of APF/CBP are unaffected by endoglycosidases F or H and contain no sugars (asdetermined on gas-capillary chromatography after acid hydrolysis) and consequently do not bind to concanavalin A (conA) or other lectins (Kannicht C, et al., Manuscript submitted, 1992). Nonetheless, when untreated bile is applied to a conA column, a proportin of APF/CBP is retained and removed subsequently by the specific eluant, a-methyl mannoside (Lafont H, Personal communication, 1992). This behavior may be due to binding of APF/CBP to the glycoproteins and amphiphilic compounds that adsorb directly to the conA. Other investigators have usually not detected APF/ CBP in bile, conA-adsorbed fractions of bile or biliary vesicles. This is due mainly to the low molecular weight (7 kD) of APF/CBP, which is lost, therefore, by ultrafiltration and dialysis through devices with pores that allow passage of proteins of 10 kD or less, by running off the ends of gels prepared with less than 12%acrylamide, by elution from the gel during the customary prolonged fixation with poorly polar solvents and by staining gels with Coomassie blue only and not with silver. APF/CBP is detected in all biles, whether or not gallstones are present, with attention to these precautions. The study protocol used by Kestell et al. (1) parallels methods used initially by Shimizu et al. (4,5)of isolation of CBP after demineralization of cholesterol gallstones and after precipitation from bile by addition of 0.5 m o m CaC1, (29-31). There are, however, two important differences in the procedures of Kestell et al.: the first is the apparent omission of protease inhibitors during the EDTA extraction, which are considered essential in the isolation of proteins from biomineralization systems (35). The second difference is the omission of the Sephadex G-25 gel filtration of the EDTA extract, which removes most of the lipids and other small molecules, including bile pigments, bile salts, peptides and amino acids (4,5,31),as well as calcium and EDTA. The second difference is especially important. Isolation of CPB by PAGE in a 12% acrylamide gel cannot yield pure material because the 6.5-kD peptide runs at the gel front and may contain other molecules of molecular weights less than 12 kD that also run at the front. The presence of these impurities likely accounts for major differences between their amino acid analyses and more recent preparations from other laboratories (30, 31) in which 15% to 16.5% acrylamide gels were used to purify APF/CBP and remove smaller contaminants not separated on the 12%gels, including bile pigments, bile salts, oligopeptides and amino acids. Because lipids were not removed, it is probable that Kestell et al. did not isolate APF/CBP protein but rather
HEPATOLOGY
something akin to the bile pigment-lipoprotein complex ( 2 , 3 )and biliary vesicles (36,37).The particle size of 60 nm detected on quasielastic light scattering and the minute proportion ( < 4%) of protein indicate that the isolated material is likely a lipoprotein particle (l), though the researchers inexplicably failed to analyze for lipids. Unfortunately, Kestell et al. therefore cannot attribute the observed properties of their isolated material (self-aggregation,pigment binding, calcium binding and inhibition of CaCO, precipitation) to the protein per se; rather, these might be properties of the presumed lipid components, of the bound bile salts or bile pigments or of the whole complex. For example, glycoconjugated, dihydroxy-bile salts, even at premicellar concentrations, inhibit the crystallization of calcium carbonates (38) and phosphates (39,401. Kestell et al. (1) confirm the prior reports of the presence of APF/CBP in gallbladder and hepatic bile and cholesterol and pigment gallstones, its precipitation by calcium and its binding of lipids. The major new findings are the first direct demonstrations of calcium binding by the “protein” and its strong tendency to self-aggregate, especially in the presence of calcium. As noted by the authors, however, this self-aggregation and precipitation of the lipoprotein particles precluded determination of the affinity constants for binding of calcium. Their dialysis studies have two other limitations: (a) the continued slow decline in 45Ca in the bath and the irregularity of the counts in the contents of the dialysis sac suggest that equilibrium was not attained, an event that takes many hours when precipitates are present or forming in the system (41); and (b) the failure to measure APF/CBP in the bath makes the claim that the bath was protein free inappropriate. Dialysis with a 10-kD cutoff membrane would likely allow some of the 6.5-kD APF/CBP monomer to enter the dialysate. How Might APFICBP Regulate Calcium Salt Precipitation, and Why Is I t Present in Gallstones?We believe that APF/CBP is the small regulatory polypeptide of the biomineralization system in bile (4,6,42).APF/CBP has characteristics typical of peptides that regulate calcium deposition in bones and teeth (35, 42, 43) and in renal (44) and pancreatic (45) calculi: size less than 20 kD, amphipathic and anionic character and phosphorylated serines. We propose that APF/CBP binds to the hydrophobic, unglycosylated “intermediate piece” of the large structural protein, mucin (46),and then binds Ca2 ions to its own anionic groups, which are properly spaced to act as a template for growth of the calcium salt crystals (6). In analogy to a building, mucins are the girders, APF/CBP is the mortar and the calcium salt crystals are the bricks. As the crystal grows on the matrix, more APF/CBP (by way of its anionic domains) may bind to the growth face of the calcium salt crystal. Depending on conditions, the exposed hydrophobic domain of APF/CBP could then (a) arrest further accretion of calcium salts to the growing crystal; (b) bind cholesterol, resulting in a cholesterol stone; or (c) bind more mucin, triggering a new cycle of mineralization in a new layer of the calculus. APF/CBP, like other small molecules, might diffuse into the interstices of the already formed stone (47). +
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Vol 16. No. 6. 1992
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Using MAbs for ELISA, it was determined that con- specifically with cy-methyl mannoside appears to reside centrations of APF/CBP in normal bile average 1.0 mainly in the 5-kD rather than in the 130-kD protein mg/ml (gallbladder) and 0.4 mg:’ml (hepatic);concentra- (52). Because the 5-kD protein reacts with MAbs tions relative to bile salts average 17% higher in bile prepared against APF, this protein is probably APF/CBP from patients with cholesterol gallstone5 and 29% or a large fragment thereof (Lafont H, Personal comhigher in those with pigment gallstones (Doming0 N , et munication). Clearly APF/CBP can affect precipitation al., Manuscript submitted, 1992 ) . These relatively small from bile of cholesterol and of calcium salts. Future Directions. It remains to characterize fully the increases in APF/CBP concentrations in the bile of patients with gallstone suggest that an intrinsic qualitative molecular structure of this protein, determine whether change in the protein might be found in patients with this structure is altered qualitatively in the bile of gallstone disease, akin to abnormalities in urinary neph- patients with gallstones and elucidate the mechanisms by which APF/CBP affects calcium and cholesterol rocalcin in patients with urinary tract stones (44). Role of APFICBP in Lipid Transport and Cholesterol precipitation, quality of biliary vesicles and hepatocytic Precipitution. MAbs against the N-terminal and transport of cholesterol. This includes determination in C-terminal portions (but not against the intervening model systems of whether the bound lipids, bile salts, polypeptide) of ApoA-I cross-react with APFCBP ( 31 !. bile pigments and calcium are essential to or modulate Because APF/CBP contains three amino acids and six the actions of APF/CBP on cholsterol and calcium epitopes that are not found in ApoA-I, the former cannot precipitation. Because the APF/CBP extracted from be a fragment of the latter. These two proteins are gallstones may be partly degraded during the long probably different products of a common supergene, sojourn in the gallbladder or during the extraction generated by alternative splicing of messenger RNA of the minerals from the protein, further studies transcribed by the gene. Six MAbs prepared against should use preferentially the protein isolated freshly APF, half of which also cross-react with Apo A-I, react from bile and compare APF/CBP from normal and also with epitopes on the surface of HDL3 t 3 1 ~ ,sug- abnormal biles. Peptide maps (fingerprints) and amino gesting that APF is partially exposed on the surface of acid sequences of APF/CBP preparations are needed but this plasma lipoprotein particle. Specific binding sites for have thus far been unsuccessful because of a blocked APF are present on the basolateral plasma membranes N-terminal amino acid. Ultimately, the identity and of rat hepatocytes (481, further suggesting a role for APF structure of APF/CBP and possible alterations in pain lipoprotein uptake. Indeed, hepatocytic uptake, intra- tients with gallstones will be established by sequencing cellular distribution and metabolism of cholesterol are the gene(s1. modified by addition of APF to infused liposomes containing cholesterol (49). J. DONALDOSTROW,M.D. Immunoelectron microscopy has localized APF,’CBP D.V.A. Lakeside Medical Center to the endoplasmic reticulum of rat hepatocytes but not Chicago, Illinois 60611 bile duct or gallbladder epithelia; APFKBP is synthesized by isolated rat hepatocytes (50).L k e the biliary REFERENCES lipid particles with which it is associated, hepatic secretion of APF/CBP in rats is regulated by the rate of Kestell MF. Sekijima J , Lee SP, Park HZ, Long M, Kaler EW. A secretion and hydrophobicity of secreted bile salts ( 511. calcium binding protein in bile and gallstones. HEPATOLOW 1992;16:1315-132i . The dissociation between biliary secretion of APF and Nalbone G. Lafont H. Vime JL. Domineo N. Lairon D. Chabert C. lipids when dehydrocholate is infused (51~ 1 . however, is Lechene P: et al. The apoprotein fraction of the bile lipoprotein not in agreement with the concept that APFiCBP itself complex: isolation, partial characterization and phospholipid regulates biliary lipid secretion. binding properties. Biociumie 1985:61:1029-1041. Martigne M, Domingo N. Lafont H, Nalbone G, Hauton JC. Because APF/CBP is the major apolipoprotein of the Purification of the human anionic polypeptide fraction of the bile pigment-lipoprotein complex ( 2 , 3 , . we were not apo-bile lipoprotein complex by zonal ultracentrifugation. Lipids surprised to find that APFCBP inserts itself into 1985;20:884-889. cholesterol-lecithin vesicles, causing their permeabilShimizu S, Sabsay B. Veis A, Ostrow JD. Rege RV, Dawes LG. ization, aggregation and fusion and promoting growth Isolation of an acidic protein from cholesterol gallstones which inhibits the precipitation of calcium carbonate in vitro. J Clin of cholesterol crystals in supersaturated bile (33).These Invest 1989;84:1990-1996. effects are augmented by addition of 10 mmol/L calcium Oludo M. Shimizu S, Ostrow JD. Nakayama F. Isolation of a chloride, possibly because the hydrophobic portion of calcium-regulatory protein from black pigment gallstones: simiAPF/CBP inserts itself into the electrically neutral larity with a protein from cholesterol gallstones. HEPKroLOGY 1992;15:1079-1085. lecithin-cholesterol vesicles of bile, with its anionic Ostrow JD. Unconjugated bilirubin and cholesterol gallstone groups at the vesicular surface available to react 1990;12(suppl 1 1219S-226S. formation. HEPATOLOGY bivalently with Ca2 + ( 12 1. These preliminary studies. Ostrow JD,Celic L. Apparent solubility products (K”sp) of the however, must be confirmed in experiments with calcium salts of unconjugated bilirubin (B) indicate supersatulower, more physiological concentrations of Ca2 ration of human gallbladder bile with Ca(HB),, but not CaB 1 Abstractl. HEPATOLOGY 1987;7:1111. ( 5 2 mmol/L), pretreatment of the protein with Chelex Vadali M, Ostrow JD. Mukerjee P, Celic L. Aqueous solubility to remove all Ca2 from control samples and additional products of calcium bilirubinate salts, determined by solvent controls containing added calcium but no protein. partition from chloroform into buffer aqueous solutions of CaCI,. Interestingly, the pronucleating activity for cholesterol In: Proceedings of the XI1 International Bile Acid Meeting. Basel: 1992. of the fraction of bile that is retained by conA and eluted +
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9. Shiffman ML, Sugerman HJ, Moore EW. Human mucosal gall- 32. Martigne M, Domingo N, Lechene de la Porte P, Lafont H, Hauton JC. Identification and localization of apoprotein fraction of the bile bladder function: effect of concentration and acidification of bile on lipoprotein complex in human gallstones. Scand J Gastroenterol cholesterol and calcium solubility. Gastroenterology 1990;99: 1988;23:731-737. 1452-1459. 10. Knyrim K, Vakil N. The effects of synthetic human secretin on 33. Domingo N, Groen B, Lechene P, de Bruijn M, Ostrow JD, Lafont H. Anionic polypeptide fraction (APF) avidly interacts with calcium carbonate solubility in human bile. Gastroenterology cholesteroMecithin vesicles and promotes crystal growth in model 1990;99:1445-1451. bile [Abstract]. Gastroenterology 1992;102:A802. 11. Carey MC, Small DM. The physical chemistry of cholesterol solubility in bile: relationship to gallstone formation and disso- 34. Trotman BW, Rajagopalan C, Bernstein SE. Monoconjugated bilirubin is a major component of hemolysis-induced gallstones in lution in man. J Clin Invest 1978;61:998-1026. 1988;8:919-924. 12. Moore EW. Biliary calcium and gallstone formation. HEPATOLOGY mice. HEPATOLOGY 35. Veis A. Bones and teeth. In: Piez KA, Reddi H, eds. Extracellular 1 9 9 0 ; 1 2 ( ~ ~ p1):206S-218S. pl matrix biochemistry. New York: Elseviermorth Holland Pub13. Crowther RS,Soloway RD. Pigment gallstone pathogenesis: from lishers, 1984:329-374. man to molecules. Semin Liver Dis 1990;10:171-180. 14. Holzbach RT. Recent progress in understanding cholesterol 36. Somjen GJ, Gilat T. A non-micellar mode of cholesterol transport in bile. FEBS Lett 1983;156:265-268. crystal nucleation as a precursor to human gallstone formation. 37. Ulloa N, Garrido J , Nervi F. Ultracentrifugal isolation of vesicular HEPATOLOGY 1986;6:1403-1406. carriers of biliray cholesterol in native human and rat bile. 15. Holzbach RT, Kibe A, Thiel E, Howell JH,Marsh M, Hermann RE. HEPATOLOGY 1987;7:235-244. Biliary proteins: unique inhibitors of cholesterol crystal nucle38. Marteau C. Portugal H, Pauli AM, Gerolami A. Effect of ation in human gallbladder bile. J Clin Invest 1984;73:35-45. glycoursodeoxycholate on precipitation of calcium carbonate. 16. Burnstein JM, Ilson RG, Petrunka CN, Strasberg SM. Evidence HEPATOLOGY 1985;5:1209-1212. for a potent nucleation factor in gallbladder bile of patients with 39. Van der Meer R, DeVries HT. Differential binding of glycine- and cholesterol gallstones. Gastroenterology 1983;85:801-807. taurine-conjugated bile acids to insoluble calcium phosphate. 17. Levy PF, Smith BF, Lamont JT. Human gallbladder mucin Biochem J 1985;229:265-268. accelerates in uitro nucleation of cholesterol in artificial bile. 40. Qui SM, Wen G, Hirakawa N, Soloway RD, Hong NK, Crowther Gastroenterology 1984;87:270-275. RS. Glycochenodeoxycholic acid inhibits calcium phosphate pre18. Groen AK, Noordam C, Drapers JAG, Egbers P, Jansen PLM, cipitation in vitro by preventing the transformation of amorphous Tytgat GNJ. Isolation of a potent cholesterol nucleationcalcium phosphate to calcium hydroxyapatite. J Clin Invest promoting activity from human gallbladder bile: role in the 1991;88:1265-1271. pathogenesis of gallstone disease. HEPATOLOGY 1990;11:525-533. 1984;4(suppl): 19. Harvey PRC, Upadhya GA, Strasberg SM. Immunoglobulins as 41. Nancollas GH. Crystallization in bile. HEPATOLOGY 169s-172s. nucleating proteins in the gallbladder bile of patients with 42. Veis A, Sabsay B. Bone and tooth formation: insights into cholesterol gallstones. J Biol Chem 1991;266:13996-14003. mineralization strategies. In: Westbroek P, DeJong EW, eds. 20. Groen AK. Nonmucous glycoproteins as pronucleating agents. Biomineralization and biological metal accumulation: biological HEPATOLOGY 1990;12(suppl 1):189S-l94S. and geological. Dordrecht, The Netherlands: D. Riedel Co., 21. Busch N, Holzbach RT. Crystal growth-inhibiting proteins in bile. 1982~273-284. HEPATOLOGY 1990;12(suppl 1):195S-l99S. 22. Schwarzendrube J , Nuutinen H, Abei N, Chandler K, Williams C, 43. Sabsay B, Stetler-Stevenson WG, Lechner JH, Veis A. Domain structure and sequence distribution in dentin phosphophoryn. Holzbach RT. Amino acid sequencing and immunological localBiochem J 1991;276:699-707. ization of a unique 120 kD dimeric crystal growth-inhibiting biliary glycoprotein [Abstract]. Gastroenterology 1992;102:A882. 44. Nakagawa Y, Ahmed M, Hall SL, DeGanello S, Coe FL. Isolation from human calcium oxalate renal stones of a glycoprotein 23. Gallinger S, Harvey PRC, Petrunka CN, Strasberg SM. Effect of inhibitor of calcium oxalate crystal growth: evidence that nephbinding of ionized calcium on the in vitro nucleation of cholesterol rocalcin from patients with calcium oxalate nephrolithiasis is and calcium bilirubinate in human gallbladder bile. Gut 1986;27: deficient in y-carboxyglutamic acid. J Clin Invest 1987;79:17821382-1386. 1787. 24. Shermak MA, Lipsett PA, Kaufman HS, Fox-Talbot MK, Pitt HA, Lillemoe KD. Calcium interacts with biliary proteins to induce 45. DeCaro A, Multigner L, Lafont H, Lombard0 D, Sarles H. The molecular characteristics of a human pancreatic acidic phosphocholesterol crystal nucleation [Abstract]. Gastroenterology 1992; protein that inhibits calcium carbonate crystal growth. Biochem J 102:A941. 1984;222:669-677. 25. Sutor DJ, Percival JM. Presence or absence of inhibitors of crystal growth in bile. 1. Effect of bile on the formation of calcium 46. Smith BF, Lamont JT. Hydrophobic binding properties of bovine gallbladder mucin. J Biol Chem 1984;259:12170-12177. phosphate, a constituent of gallstones. Gut 1976;17:506-510. 26. Sutor DJ, Percival JM. The effect of bile on the crystallization of 47. Sanabria JR,Upadhya GA, Harvey PRC, Strasberg SM. Diffusion of substances into cholesterol gallstones [Abstract]. Gastroentercalcium carbonate. Clin Chim Ada 1978;89:479-484. ology 1992;102:A878. 27. Sciaretta A, Ligabue A, Garuti G, Pieromaldi S, Verri I, Giacobazzi G, Malaguti P. Inhibitory activity of gallbladder bile on calcium 48. Martigne M, Meli B, Mahlberg F, Domingo N, Chanussot F, Lafont H, Hauton JC. Detection and characterization of anionic polypepcarbonate crystallization in vitro: a comparison between normal tidic fraction binding sites in rat liver plasma membranes and subjects and gallstone patients. Scand J Gastroenterol 1984;19: cultured hepatocytes. Biochim Biophys Acta 1989;979:341-346. 626-630. 28. Dawes LG, Moore EW, Rege RV, Shimizu S, Ostrow JD. Canine 49. Martigne M, Domingo N, Chanussot F, Nalbone G, Lafont H, Hauton J . Effects of bile anionic polypeptide fraction on the fate bile contains anticrystallization factors that inhibit precipitation of cholesterol carried by liposomes in the rat. Proc Soc Exp Biol of calcium carbonate. HEPATOLOGY 1991;14:701-706. Med 1988;187:229-234. 29. Shimizu S, Sabsay B, Veis A, Ostrow JD, Dawes LG, Rege RV. A 12-kDa acidic protein from cholesterol gallstones inhibits precip- 50. Domingo N, Botta D, Martigne-Cros M, Lechene de la Porte P, Pak-Leung P, Hauton J , Lafont H. Evidence for the synthesis and itation of calcium carbonate and is present in human bile secretion of APF - a bile lipid associated protein - by isolated rat [Abstract]. HEPATOLOGY 1988;8:1257. hepatocytes. Biochim Biophys Acta 1990;1044:243-248. 30. Lafont H, Ostrow JD, Shimizu S,Domingo N, Lechene P, Hauton JC, Rege RV, et al. Are anionic polypeptide fraction (APF) from 51. Chanussot F, Domingo N, Tuchweber B, Lafont H, Yousef IB. Influence of dehydrocholic and cholic acids infusions on the biliary bile and calcium-binding protein (CBP) from gallstones identical secretion of anionic polypeptide fraction, the major apoprotein of [Abstract]? Gastroenterology 1991;100:A764. the biliary lipoprotein complex. Scand J Gastroenterol 1992;27: 31. Domingo N, Grosclaude J , Bekaert ED, Mege D, Chapman MJ, 238-242. Shimizu S, AyraultJanier M, et al. Epitope mapping of the human biliary amphipathic anionic polypeptide (APF): similarity 52. DeBruijn MAC, Mok K, Doming0 N, Lafont H, Tytgat GNJ, Groen AK. Further characterization of the cholesterol crystallization with a calcium-binding protein (CBP) isolated from gallstones and promoting lipoprotein in biliary Con-A-binding protein [Abbile, and immunological cross-reactivity with apolipoprotein A-I. stract]. Gastroenterology 1992;102:A310. J Lipid Res 1992;33:1419-1430.
