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Composition and immunofluorescence studies of biliary “sludge” in patients with cholesterol or mixed gallstones
Composition and immunofluorescencestudies of biliary %ludge”in patients with cholesterol or mixed gallstones Paulette
Lechene
de la Porte,
Huguette
Lafont,
Nicole
Benedikta
Ztindt*
and Dieter
Background/Aims: Gallbladder bile from patients with cholesterol or mixed gallstones frequently contains biliary “sludge”, a suspension of cholesterol monohydrate crystals and pigment granules embedded in mucin and proteins. The composition of biliary “sludge” and the preferential localization of mucin and proteins could be an indicator for its potential role in gallstone formation. Methods: Ultracentrifugation (100 000 g/l h) was used to precipitate “sludge” from bile, and the concentration difference of its main components between native bile and ultracentrifuged bile samples was calculated. After purification of the sediment, immunolocalization was performed for the detection of mucin, IgA, albumin, aminopeptidase, and anionic polypeptide fraction using polyclonal and monoclonal antibodies. Results: The amount of sludge in gallbladder bile was 4.26 mg/m1?0.78 (mean+SEM) in patients with cholesterol and 2.51 mglml-cO.39 in patients with
A
number of biliary proteins have been suggested to play a role in the pathogenesis of cholesterol gallstones (l-7). Some were identified as pronucleating factors like mucin, IgA or aminopeptidase N (ApN) (S-18), and others as antinucleating factors like the anionic polypeptide fraction (APF), calcium binding protein (CBP), Apo Al or apo AII, (1926). Most of these data concern in vitro studies in model bile systems. In a recent study, immunolocalization of biliary proN INCREASING
Received 28 October 1999; revised 4 Fehrunry:
accepted 15 Frhrucvy 2000
Correspondence: Paulette Lechene de la Porte, Unite 476, INSERM, Nutrition Humaine et Lipides, 18, avenue Mozart, 13009 Marseille, France. Tel: 33 491 758600. Fax: 33 491 751562.
352
Domingo.
Gunther
Meyer’,
Iris Mtiller*,
Jiingst’
mixed stones and cholesterol was the main component (48.9*4.6’% and 44.427.1s). The sediment appeared as a mixture of vesicular aggregates and pigment particles which were linked by a gel matrix of mucin containing cholesterol crystals. While anionic polypeptide fraction and aminopeptidase were associated to pigments, TgA was uniformly spread in the crystalline parts of “core-like” structures, and albumin, when it was present, appeared as randomly located small spots. Conclusions: Our study demonstrates that the cholesterol content and the distribution pattern of mucin and different proteins is similar in the sediments of biliary “sludge” to that previously shown in cholesterol and mixed gallstones. This suggests that biliary “sludge” represents an early stage of gallstone formation in these patients. Key words: Bile; Cholesterol; Composition; fluorescence; Mucin; Proteins: Sludge.
Immuno-
teins in cholesterol rich gallstones showed that mucin is specifically associated with cholesterol crystals; APF is associated with pigment deposits and ApN displays the same localization was APE IgA and albumin, when they are present, show no specific localization in the stones (13). Gallbladder bile from cholesterol gallstone patients frequently contains biliary “sludge”, a suspension of cholesterol monohydrate crystals or calcium bilirubinate granules embedded in mucus, a mixture of mucin and proteins (27,28). Gallbladder mucin hypersecretion and accumulation in the gallbladder as a viscoelastic gel precedes the formation of gallstones in both animal (29-31) and human studies (2,32). Moreover, some investigations have shown that the initial nucleation of cholesterol crystals
Irnmunofhorescence
occurs in the gallbladder mucus gel (29,30,33). Although, mucin is supposed to be an important pronucleating factor in supersaturated native (34) and model biles (9,10,12,35), the mechanisms which lead to an accelerated cholesterol monohydrate crystal formation are still unknown. Each compound found in gallstones must have precipitated from bile. This may occur only if the concentration of this element is in a thermodynamically supersaturated range. A number of studies suggest that cholesterol/phospholipid vesicles in the gallbladder bile favor cholesterol crystal formation (36-38). Biliary proteins were demonstrated to play a role in calcium and probably cholesterol precipitation by destabilizing these vesicles. APF is found in the vesicular fraction and was shown to stabilize aggregated vesicles and to protect them against the destabilizing effect of calcium (24). This is probably due to its phospholipid binding properties (39). Moreover, the stability of these vesicles can also be modulated by mucin, whose lipid-binding properties are of considerable interest (10,12,40). Mucin is known as a nucleating protein and stimulates the crystal growth in supersaturated model biles (12,14). The nucleating action of gallbladder mucin (GBM) probably depends on hydrophobic binding sites on the non-glycosylated portion of its peptide core as proteolytic digestion impairs both lipid binding and nucleating activity (35,40). In gallstones, the cholesterol crystals and calcium salts are organized on a network of organic matrix (1). It consists mainly of a polymerized mucus gel which is linked with other proteins: the small APF/CBP which have the same aminoacid composition and the larger ApN and IgA (13). Our aim was to study the amount of sludge and its main components in gallbladder bile of patients with cholesterol and mixed stones. Moreover, supposing that biliary sludge could constitute an early stage of gallstone formation, we have studied the immunolocalization of several biliary proteins in biliary sludge of these patients and compared our results to those previously described for the same proteins in cholesterol or mixed gallstones (13).
Materials and Methods Patients and collection of bile Sixty-five patients, 48 women and 17 men who underwent laparoscopic surgery because of symptomatic cholesterol or mixed gallstone disease, were included in the study. Gallstones were visualized by ultrasonography and reasonable functioning of the gallbladder was confirmed by an emptied volume of at least 30% of the fasting volume after application of a liquid test meal. It was the purpose of our study to investigate biliary “sludge” in the majority of patients with cholesterol or mixed stones. Therefore, we chose a comparatively low cut-off value of an emptied volume of 30% of the total fasting volume. Due to this selection criterion our study population comprised patients with normal and slightly impaired gallbladder function. These
studies of biliary sludge
represent about 80% of patients with symptomatic gallstones prior to elective cholecystectomy. All patients gave informed consent after a detailed explanation of the procedure to collect bile intraoperatively. During laparoscopic surgery the gallbladder was punctured and a flexible probe with side ports was inserted; bile was aspirated as completely as possible because of the known stratification of human gallbladder bile (41). Stones were removed, washed with distilled water, dried, weighed and ground to a powder. The cholesterol content of the stones was measured chemically after extraction with organic solvent and expressed as a percentage of dry weight (42). Informed consent was obtained from each patient and the study protocol conforms to the ethical guidelines of the Declaration of Helsinki as reflected in a priori approval of the ethical committee of Ludwig-Maximilians University, Munich. For ethical reasons we were not able to collect adequate bile samples in a control group, i.e. subjects with a high risk of gallstone development, like obese patients. Analysis of biliary sludge For the analysis of biliary sludge, duplicate aliquots of native and ultracentrifuged gallbladder bile were stored at -30°C prior to determination. Cholesterol was determined calorimetrically with the Liebermann-Burchard reaction after double extraction of 1 ml methanolic bile sample with petroleum ether (43). Total bilirubin was measured with an enzymatic assay using bilirubin oxidase (44). Total protein was analyzed using the Lowry assay after purification of biliary proteins (5). Quantification of mucin was performed with a modification of the method of Pearson et al. (45). Briefly, 1 ml of gallbladder bile was dissolved in Tris-buffer pH 7.4 with KSCN and taurocholic acid and incubated at 4°C for 24 h. After centrifugation 1 ml of the supernantant was separated over a Sepharose-4B-Cl column using PBS taurocholic buffer (10 mM) as the eluant. The higher molecular glycoprotein-fractions were pooled and dialysed against distilled water for 24 h to remove the taurocholic acid. The mucin concentration of the dialysed glycoprotein fraction was measured using the PAS method. Immunolocalization of proteins in sediments of gallbladder biles After the collection, 4 ml of gallbladder bile were centrifuged in polycarbonate tubes with a 50 Ti rotor at 37°C for 1 h at 100 000 g in a Beckman L5-65 ultracentrifuge (Beckman Instruments, Fullerton, CA, USA) to precipitate biliary sediments. The supernatant was microscopically free of cholesterol crystals. Protocol 1: About 0.5 mm3 of the sediment was collected with a needle, transferred to a thermanox (Nunc, Inc. Naperville, IL, USA) cover slip and covered with a small drop of gelatin-NaCl(37”C). After cooling, the sediment elements were trapped into the gelatine. The samples were then fixed for 1 h at 4°C in paraformaldehyde 4% (WI V) in PBS, then rinsed and stored in PBS overnight at 4°C before immunolocalization. Protocol 2: About 0.5 mm3 of the sediment was transferred to the bottom of a millicell-PC insert (Millipore) and immediately fixed for 1 h at 4°C in 4% paraformaldehyde in PBS. Then the specimens were rinsed through several washes with PBS. After both protocols the immunolocalization was performed by incubating the samples for 2 h at 20°C with GBM 59 mouse monoclonal antibody (IgM) against polymucin 1:400 (45), rabbit anti-human albumin 1:lOO (Nordic, Tilburg, NL), rabbit anti-human IgA 1:200 (Nordic, Tilburg, NL), rabbit anti-human APF 1:400 or rabbit anti-human anti-aminopeptidase-N 1:400 (gift from AK Groen). Non-immune rabbit serum 1:200 (Nordic, Tilburg, NL) was used as control. After several washes in PBS the specimens were reacted for 1 h at room temperature with an appropriate secondary antiserum (Jackson, Immuno Research Laboratories, Inc. West Grove, USA). Mouse monoclonal antibodies were detected with goat-anti-mouse IgM- or IGG Texas Red labeled sera and rabbit polyclonal IgG were detected with goat-anti-rabbit IgG FITC labeled serum. The secondary antisera were diluted 1:250 in PBS and were allowed
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P. Lechene de la Porte et al. to react for 1 h at room temperature. After rinsing with PBS small fragments of the sediment treated according to protocol 1 were gently isolated with a scalpel blade from the gelatine support. Such treated sediments remained semi-liquid even after the fixation procedure; they were crushed gently between slide and glass cover slip, and were immediately observed. Following protocol 2, the membrane at the bottom of the insert was delicately isolated from its support and placed between slide and cover slip. Observations were performed with a Dialux microscope (Leica) equipped for epi-fluorescence studies.
Statistical analysis Results are presented as group means and standard deviation of the mean (SEM). Group mean differences were compared by MannWhitney U-test. Discrete variables were tested by Pearson Chi-square test. Double-side p-values were reported. P-values of less than 0.05 were regarded as statistically significant.
Results Morphological aspects of the sediments After ultracentrifugation the sediments of gallbladder bile contained more or less prominent brown pigment precipitates (Fig. I) embedded in a slimy matrix of granular structures. Two kinds of cholesterol crystals were included in this matrix: classical monohydrate plates (Fig. 1) and/or tubular structures. In some patients, structures very similar to those which constitute the central core of mixed stones were observed in the sediments (Fig. 2): cholesterol crystals more or less associated with pigments radiated from a central hardly pigmented area. In other structures (Fig. 3) alternating deposits of pigment and large cholesterol layers looked like the core of gallstones.
Fig. 1. Morphological aspect of a sediment of gallbladder bile after ultracentrtfugation: pigment precipitates (P) are included in a granular matrix (M); cholesterol crystals (*) in the granular matrix are never associated with pigments (bar 1 cm=125 pm).
Immunolocalization ofproteins The Texas-Red fluorescence specific for mucin immunofluorescence was widely distributed on the microscopic field. It was associated with a dense non-amorphous matrix in which cholesterol crystals were juxtaposed (Fig. 4a & 4b). The crystals by themselves were generally not fluorescent; only a few of them were underlined by a faint red fluorescence (Fig. 4b). Mucin was mainly localized in areas rich in cholesterol crystals and seemed to be excluded from the pigment precipitate content; however, large pigment deposits were often covered with a layer of mucin. Using protocol 2, it was possible in some areas to identify clusters of vesicles (Fig. 5a) which were clearly associated with mucin (Fig. 5b). APF was clearly associated with the pigment precipitates and the specific fluorescence was stronger at the periphery of these precipitates (Fig. 6a & Fig. 7).
Fig. 2. Radiated structures, similar to the central core of mixed gallstones, containing pigment and cholesterol crystals (bar I cm=125 pm).
Fig. 3. Crystalline structures, similar to the luminuted periphery of gallstones (bar 1 cm= I25 pm).
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Immunofluorescence studies of biliary sludge
Fig. 4. Immunolocalization of gallbladder mucin in a sediment of gallbladder bile after ultracentrtfiigation. a) Normal light: cholesterol crystals: plates (*) and tubules (arrowheads), are associated with a granular matrix. b) Fluorescent light: Mucin is located in the granular matrix (A4), only a few cholesterol crystals (arrowheads) are underlined by the Texas-Redjuorescence (bar I cm=125 ,um).
Cholesterol crystals and the matrix were never labeled (Fig. 6b). The aminopeptidase-N was also associated with the pigment precipitates (Fig. 8) and showed the same localization as APE Albumin was scarcely detected in the sediments, but
Fig. 5. a) Lipid vesicle aggregates (arrowheads) in a sediment of gallbladder bile after ultracentrtfugation. b) Lipid vesicles aggregates are clearly labeled by the Texas-Red fluorescence for gallbladder mucin (bar I cm=125 ,um).
if present albumin appeared as little spots which were randomly located. No specific localization was detected for IgA, except in the structures, which looked like the core of gallstones; in this case IgA seemed to be associated with the crystalline part of these structures. 35.5
I? Lechene de la Porte et al. TABLE
1
Concentration difference (meantSEM) of cholesterol, mucin. protein and bilirubin between native and ultracentrifuged gallbladder bile of 49 patients with cholesterol and 16 patients with mixed stones. The percentage of the major components of biliary “sludge” in gallbladder bile is shown in parentheses Cholesterol
Cholesterol Mucin Protein Bilirubin Total sludge quantity
stones
Mixed stones
mg/ml
0%
mg/ml
‘%I
2.08-cO.39 0.41 to.20 1.70t0.51 0.07~0.01 4.2620.78
(48.924.6) (9.623.0) (4O.Oi7.2) (2.711.1)
1.11t0,23 0.15ro.08 1.08t0.20 0.1610.08 2.51 -to.39
(44.427.1) (8.4t3.2) (43.5f4.9) (6.5+1.8)
Quantification of biliary sludge As a result of cholesterol analysis in the collected gallstones, 49 patients (38 women and 11 men) were classified as cholesterol stone (>50’%) cholesterol) carriers. Sixteen patients (10 women and 6 men) had intermediate results (lo--50% cholesterol) and represented the mixed stone group. The major components of biliary sludge, i.e. cholesterol, protein, mucin and bilirubin, were determined as the concentration difference (mg/ml) between native and ultracentrifuged gallbladder bile. The results for the two stone groups are given in Table 1. The findings demonstrated no significant differences in the total amount or the single components of biliary sludge between the two groups of patients. This was due to a wide variation in amounts of sludge in gallbladder bile samples as determined by the ultracentrifugation technique. The calculation derives partially from basal values in native and ultracentrifuged gallbladder bile which have been presented previously (46). The percentages of cholesterol in biliary sludge in the two groups of patients were similar to the percentage of cholesterol in cholesterol or mixed gallstones. These findings and the immunofluorescence studies suggest that biliary “sludge” is a necessary intermediate stage of gallstone formation in these patients.
Discussion Fig. 6. Immunolocalization of APF in a sediment ofgullbladder bile after ultracentrifugution. u) Normal light. Cholesterol crystal (*) und pigment precipitates (P) are embedded in a mucous matrix (M). b) After protocol 2, APF was shown to he associated to the pigment aggregates (P); cholesterol crystals (*) are never labeled by the immunoreaction (bur I cm=125 pm).
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Gallbladder bile from gallstone patients contains nonsoluble material which comprises a suspension of cholesterol crystals and/or calcium bilirubinate granules embedded in mucin, a mixture of high molecular weight mucus glycoprotein with a low mannose content and proteins (27,28). In the presented study, “sludge” was defined as any non-soluble material in bile which precipitates after 1 h of ultracentrifugation. Instead of centrifugation at
Immunofuorescence
Fig. 7. Irnmunolocalization of APF in a sediment of gallbladder bile after ultracentrifugation after the protocol 1. the specific FITC yellow fluorescence is clearly associated to the periphery of thepigmentprecipitates (*) (bar 1 cm= 500 pm).
3000 g we used ultracentrifugation for 1 h at 100 000 g to separate “sludge” from gallbladder bile to ascertain that any residual cholesterol crystals were sedimented, providing a correct quantification of biliary sludge. In this strict and not generally accepted definition, each bile contains some amount of “sludge”. Due to methodological difficulties in analyzing the components of the sediment after ultracentrifugation an indirect approach was chosen. Measurement of the concentration differences of the main components of sludge between native bile and the supernatant after ultracentrifugation was used. This allowed the determination of comparably tiny amounts of “sludge” not visible in the gallbladder by diagnostic methods like ultrasonography. In addition, the relationship between selected biliary proteins, previously shown to be present in cholesterolcontaining gallstones, and cholesterol crystals or pigments were studied using immunofluorescence. As expected, the morphological studies showed that the pellets were rich in cholesterol crystals at different steps of their formation (47): tubules and the more
studies
of biliary sludge
Fig. 8. Immunolocalization of aminopeptidase N ment of gallbladder bile after ultracentrtfugation protocol I: the specific FITC-yellow fluorescence associated to the pigment precipitates (*) in the as for APF (compare to Fig. 7) (bar I cm=500
in a sediafter the is clearly same way pm).
stable monohydrate cholesterol plates. In all samples studied they were embedded in an uncolored mucous matrix, which also contained small granular structures. Beside these cholesterol crystals, brown pigment precipitates were more or less developed, which also were trapped in the mucous compound. Cholesterol crystals and pigment precipitates seemed to be randomly placed in the gel without any specific relationships; they only co-existed in the same medium. In some cases, structures resembling the radiated central core described in mixed gallstones were present, and cholesterol crystals and pigments were closely associated (Fig. 2). Other elaborate structures (Fig. 3) looked like the periphery of the more developed stones where pigment and cholesterol deposits alternated in concentric areas of mixed stones. These latter structures were rich in cholesterol crystals. Immunolocalization showed that the mucous granule-rich matrix contained mucin, and that cholesterol crystals were clearly associated with it. It is interesting that gallbladder mucin was also clearly associated with clusters of vesicles (Fig. 5) in such a quantity that it was detectable. Using three separate physical chemical 357
P. Lechene de la Porte et al
techniques including dynamic light scattering, transmission electronic microscopy and fluorescent biochemical assays, mucin-vesicle interactions were demonstrated already in model bile (48). Our study in native bile supports this finding that mucin induces vesicle aggregation and fusion and, by this mechanism facilitates cholesterol crystallization in bile. In cholesterol gallstones a very bright mucin signal has been described associated with cholesterol crystals but not with pigment-rich areas (13). Mucin was interlaced among the cholesterol crystals and concentrated at the boundaries between the cholesterol and pigment zones. As in stones, in sludge samples mucin surrounded both the pigmented and cholesterol zones but was never identified within the pigment deposit layers. The concept that mucin gel promotes the crystallization of cholesterol in bile was also supported by a recent study (49). The gel matrix can often become trapped within the growing crystals or between crystals and in mixed gallstones cholesterol crystals appeared to be agglomerated by a thin film of mucin (13). Mucin is also a scaffold on which smaller acidic proteins such as CBP and/or APF are bound, which in turn regulate the deposition of mineral components. In . mixed gallstones (13) APF was demonstrated only in zones which contained calcium salts and bile pigments. It was especially located at the interfaces between cholesterol and pigments deposits. In the sediments investigated we obtained the same kind of localization for APE This small polypeptide (7 kD) is highly amphipathic, leading to avid self-aggregation. It is strong phospholipid and pigment binding as well as bile salts binding (50). In model biles, APF was shown to have an inhibitory function on cholesterol crystallization by stabilizing the vesicles against the effect of calcium (51). In vitro studies with model bile revealed that APF retains cholesterol within supersaturated multivesicular aggregates and markedly decreased the rate of crystal formation when compared to the vesicles without APF (50). When calcium was present in the model bile APF counterbalanced the destabilizing effect of calcium. The crystal mass was decreased in the presence of APF but the nucleation time was not modified. Calcium has been shown to be present within cholesterol stones in several specific areas (51). The concentration of calcium in the center of stones suggested the possibility that calcium salts may act as a nidus for subsequent cholesterol crystallization and stone formation. in the presence of calcium, CBP presented a clear cooperative effect with mucin to promote and aggregate cholesterol crystals (20). In sediments as in stones, a nucleating promotor
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protein, aminopeptidase-N, was clearly associated with pigment deposits, at their junction to cholesterol at the same localization as APE Albumin was not detected in the “sludge samples” except in some cases as infrequent “spots” randomly located. Similarly, IgA, a protein widely thought to promote cholesterol crystallization showed no specific localization in the sediments or in gallstones (13). Only in a few cases was this protein clearly associated with aggregations of cholesterol crystals which resemble the concentric cholesterol layers at the periphery of stones. Interestingly, biliary secretory IgA has recently been described as a major constituent of a group of cholesterol crystal-binding proteins (53). Thus crystal-binding IgA may be an important modulator of cholesterol crystal agglomeration into stones and stone growth in viva, as supposed by Busch et al. (53). In conclusion, the striking similarities concerning the relationship between cholesterol, biliary pigments and biliary proteins in cholesterol-containing gallstones and in different solid components of the sediment in gallbladder bile suggest strongly that biliary “sludge” is a necessary intermediate step in gallstone formation.
Acknowledgements This paper is dedicated to Gustav Paumgartner for generous support and critical discussion of the subject. Parts of these results were presented at the Annual Meeting of the American Gastroenterological Association in New Orleans, May 1998 and appeared as an abstract in Gastroenterology 1998; 114: A 528.
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,
1
P. Lechene de la Porte et al. 50. Lafont H, Domingo N, Groen AK, Kaler EW, Lee SP Koehler R, et al. APFICBP the small, amphipathic anionic protein(s) in bile and gallstones, consists of lipid-binding and calcium-binding forms. Hepatology 1997; 25: 105463. 51. Halpern 2, Lafont H, Arad J, Domingo N, Peled Y, Konikoff F, et al. The distribution of the biliary anionic polypeptide fraction between cholesterol carriers in bile and its effect on nucleation. J Hepatol 1994; 21: 979.-83.
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52. Kaufman HS, Magnusson TH, Pitt HA, Frasca P, Lillemoe KD. The distribution of calcium salt precipitates in the core, periphery and shell of cholesterol, black pigment and brown pigment gallstones, Hepatology 1994; 19: 1124-32. 53. Busch N, Lammert F, Matern S. Biliary secretory immunoglobulin A is a major constituent of a new group of cholesterol crystal-binding proteins. Gastroenterology 1998; 115: 129-38.
Lechene
de la Porte,
Huguette
Lafont,
Nicole
Benedikta
Ztindt*
and Dieter
Background/Aims: Gallbladder bile from patients with cholesterol or mixed gallstones frequently contains biliary “sludge”, a suspension of cholesterol monohydrate crystals and pigment granules embedded in mucin and proteins. The composition of biliary “sludge” and the preferential localization of mucin and proteins could be an indicator for its potential role in gallstone formation. Methods: Ultracentrifugation (100 000 g/l h) was used to precipitate “sludge” from bile, and the concentration difference of its main components between native bile and ultracentrifuged bile samples was calculated. After purification of the sediment, immunolocalization was performed for the detection of mucin, IgA, albumin, aminopeptidase, and anionic polypeptide fraction using polyclonal and monoclonal antibodies. Results: The amount of sludge in gallbladder bile was 4.26 mg/m1?0.78 (mean+SEM) in patients with cholesterol and 2.51 mglml-cO.39 in patients with
A
number of biliary proteins have been suggested to play a role in the pathogenesis of cholesterol gallstones (l-7). Some were identified as pronucleating factors like mucin, IgA or aminopeptidase N (ApN) (S-18), and others as antinucleating factors like the anionic polypeptide fraction (APF), calcium binding protein (CBP), Apo Al or apo AII, (1926). Most of these data concern in vitro studies in model bile systems. In a recent study, immunolocalization of biliary proN INCREASING
Received 28 October 1999; revised 4 Fehrunry:
accepted 15 Frhrucvy 2000
Correspondence: Paulette Lechene de la Porte, Unite 476, INSERM, Nutrition Humaine et Lipides, 18, avenue Mozart, 13009 Marseille, France. Tel: 33 491 758600. Fax: 33 491 751562.
352
Domingo.
Gunther
Meyer’,
Iris Mtiller*,
Jiingst’
mixed stones and cholesterol was the main component (48.9*4.6’% and 44.427.1s). The sediment appeared as a mixture of vesicular aggregates and pigment particles which were linked by a gel matrix of mucin containing cholesterol crystals. While anionic polypeptide fraction and aminopeptidase were associated to pigments, TgA was uniformly spread in the crystalline parts of “core-like” structures, and albumin, when it was present, appeared as randomly located small spots. Conclusions: Our study demonstrates that the cholesterol content and the distribution pattern of mucin and different proteins is similar in the sediments of biliary “sludge” to that previously shown in cholesterol and mixed gallstones. This suggests that biliary “sludge” represents an early stage of gallstone formation in these patients. Key words: Bile; Cholesterol; Composition; fluorescence; Mucin; Proteins: Sludge.
Immuno-
teins in cholesterol rich gallstones showed that mucin is specifically associated with cholesterol crystals; APF is associated with pigment deposits and ApN displays the same localization was APE IgA and albumin, when they are present, show no specific localization in the stones (13). Gallbladder bile from cholesterol gallstone patients frequently contains biliary “sludge”, a suspension of cholesterol monohydrate crystals or calcium bilirubinate granules embedded in mucus, a mixture of mucin and proteins (27,28). Gallbladder mucin hypersecretion and accumulation in the gallbladder as a viscoelastic gel precedes the formation of gallstones in both animal (29-31) and human studies (2,32). Moreover, some investigations have shown that the initial nucleation of cholesterol crystals
Irnmunofhorescence
occurs in the gallbladder mucus gel (29,30,33). Although, mucin is supposed to be an important pronucleating factor in supersaturated native (34) and model biles (9,10,12,35), the mechanisms which lead to an accelerated cholesterol monohydrate crystal formation are still unknown. Each compound found in gallstones must have precipitated from bile. This may occur only if the concentration of this element is in a thermodynamically supersaturated range. A number of studies suggest that cholesterol/phospholipid vesicles in the gallbladder bile favor cholesterol crystal formation (36-38). Biliary proteins were demonstrated to play a role in calcium and probably cholesterol precipitation by destabilizing these vesicles. APF is found in the vesicular fraction and was shown to stabilize aggregated vesicles and to protect them against the destabilizing effect of calcium (24). This is probably due to its phospholipid binding properties (39). Moreover, the stability of these vesicles can also be modulated by mucin, whose lipid-binding properties are of considerable interest (10,12,40). Mucin is known as a nucleating protein and stimulates the crystal growth in supersaturated model biles (12,14). The nucleating action of gallbladder mucin (GBM) probably depends on hydrophobic binding sites on the non-glycosylated portion of its peptide core as proteolytic digestion impairs both lipid binding and nucleating activity (35,40). In gallstones, the cholesterol crystals and calcium salts are organized on a network of organic matrix (1). It consists mainly of a polymerized mucus gel which is linked with other proteins: the small APF/CBP which have the same aminoacid composition and the larger ApN and IgA (13). Our aim was to study the amount of sludge and its main components in gallbladder bile of patients with cholesterol and mixed stones. Moreover, supposing that biliary sludge could constitute an early stage of gallstone formation, we have studied the immunolocalization of several biliary proteins in biliary sludge of these patients and compared our results to those previously described for the same proteins in cholesterol or mixed gallstones (13).
Materials and Methods Patients and collection of bile Sixty-five patients, 48 women and 17 men who underwent laparoscopic surgery because of symptomatic cholesterol or mixed gallstone disease, were included in the study. Gallstones were visualized by ultrasonography and reasonable functioning of the gallbladder was confirmed by an emptied volume of at least 30% of the fasting volume after application of a liquid test meal. It was the purpose of our study to investigate biliary “sludge” in the majority of patients with cholesterol or mixed stones. Therefore, we chose a comparatively low cut-off value of an emptied volume of 30% of the total fasting volume. Due to this selection criterion our study population comprised patients with normal and slightly impaired gallbladder function. These
studies of biliary sludge
represent about 80% of patients with symptomatic gallstones prior to elective cholecystectomy. All patients gave informed consent after a detailed explanation of the procedure to collect bile intraoperatively. During laparoscopic surgery the gallbladder was punctured and a flexible probe with side ports was inserted; bile was aspirated as completely as possible because of the known stratification of human gallbladder bile (41). Stones were removed, washed with distilled water, dried, weighed and ground to a powder. The cholesterol content of the stones was measured chemically after extraction with organic solvent and expressed as a percentage of dry weight (42). Informed consent was obtained from each patient and the study protocol conforms to the ethical guidelines of the Declaration of Helsinki as reflected in a priori approval of the ethical committee of Ludwig-Maximilians University, Munich. For ethical reasons we were not able to collect adequate bile samples in a control group, i.e. subjects with a high risk of gallstone development, like obese patients. Analysis of biliary sludge For the analysis of biliary sludge, duplicate aliquots of native and ultracentrifuged gallbladder bile were stored at -30°C prior to determination. Cholesterol was determined calorimetrically with the Liebermann-Burchard reaction after double extraction of 1 ml methanolic bile sample with petroleum ether (43). Total bilirubin was measured with an enzymatic assay using bilirubin oxidase (44). Total protein was analyzed using the Lowry assay after purification of biliary proteins (5). Quantification of mucin was performed with a modification of the method of Pearson et al. (45). Briefly, 1 ml of gallbladder bile was dissolved in Tris-buffer pH 7.4 with KSCN and taurocholic acid and incubated at 4°C for 24 h. After centrifugation 1 ml of the supernantant was separated over a Sepharose-4B-Cl column using PBS taurocholic buffer (10 mM) as the eluant. The higher molecular glycoprotein-fractions were pooled and dialysed against distilled water for 24 h to remove the taurocholic acid. The mucin concentration of the dialysed glycoprotein fraction was measured using the PAS method. Immunolocalization of proteins in sediments of gallbladder biles After the collection, 4 ml of gallbladder bile were centrifuged in polycarbonate tubes with a 50 Ti rotor at 37°C for 1 h at 100 000 g in a Beckman L5-65 ultracentrifuge (Beckman Instruments, Fullerton, CA, USA) to precipitate biliary sediments. The supernatant was microscopically free of cholesterol crystals. Protocol 1: About 0.5 mm3 of the sediment was collected with a needle, transferred to a thermanox (Nunc, Inc. Naperville, IL, USA) cover slip and covered with a small drop of gelatin-NaCl(37”C). After cooling, the sediment elements were trapped into the gelatine. The samples were then fixed for 1 h at 4°C in paraformaldehyde 4% (WI V) in PBS, then rinsed and stored in PBS overnight at 4°C before immunolocalization. Protocol 2: About 0.5 mm3 of the sediment was transferred to the bottom of a millicell-PC insert (Millipore) and immediately fixed for 1 h at 4°C in 4% paraformaldehyde in PBS. Then the specimens were rinsed through several washes with PBS. After both protocols the immunolocalization was performed by incubating the samples for 2 h at 20°C with GBM 59 mouse monoclonal antibody (IgM) against polymucin 1:400 (45), rabbit anti-human albumin 1:lOO (Nordic, Tilburg, NL), rabbit anti-human IgA 1:200 (Nordic, Tilburg, NL), rabbit anti-human APF 1:400 or rabbit anti-human anti-aminopeptidase-N 1:400 (gift from AK Groen). Non-immune rabbit serum 1:200 (Nordic, Tilburg, NL) was used as control. After several washes in PBS the specimens were reacted for 1 h at room temperature with an appropriate secondary antiserum (Jackson, Immuno Research Laboratories, Inc. West Grove, USA). Mouse monoclonal antibodies were detected with goat-anti-mouse IgM- or IGG Texas Red labeled sera and rabbit polyclonal IgG were detected with goat-anti-rabbit IgG FITC labeled serum. The secondary antisera were diluted 1:250 in PBS and were allowed
353
P. Lechene de la Porte et al. to react for 1 h at room temperature. After rinsing with PBS small fragments of the sediment treated according to protocol 1 were gently isolated with a scalpel blade from the gelatine support. Such treated sediments remained semi-liquid even after the fixation procedure; they were crushed gently between slide and glass cover slip, and were immediately observed. Following protocol 2, the membrane at the bottom of the insert was delicately isolated from its support and placed between slide and cover slip. Observations were performed with a Dialux microscope (Leica) equipped for epi-fluorescence studies.
Statistical analysis Results are presented as group means and standard deviation of the mean (SEM). Group mean differences were compared by MannWhitney U-test. Discrete variables were tested by Pearson Chi-square test. Double-side p-values were reported. P-values of less than 0.05 were regarded as statistically significant.
Results Morphological aspects of the sediments After ultracentrifugation the sediments of gallbladder bile contained more or less prominent brown pigment precipitates (Fig. I) embedded in a slimy matrix of granular structures. Two kinds of cholesterol crystals were included in this matrix: classical monohydrate plates (Fig. 1) and/or tubular structures. In some patients, structures very similar to those which constitute the central core of mixed stones were observed in the sediments (Fig. 2): cholesterol crystals more or less associated with pigments radiated from a central hardly pigmented area. In other structures (Fig. 3) alternating deposits of pigment and large cholesterol layers looked like the core of gallstones.
Fig. 1. Morphological aspect of a sediment of gallbladder bile after ultracentrtfugation: pigment precipitates (P) are included in a granular matrix (M); cholesterol crystals (*) in the granular matrix are never associated with pigments (bar 1 cm=125 pm).
Immunolocalization ofproteins The Texas-Red fluorescence specific for mucin immunofluorescence was widely distributed on the microscopic field. It was associated with a dense non-amorphous matrix in which cholesterol crystals were juxtaposed (Fig. 4a & 4b). The crystals by themselves were generally not fluorescent; only a few of them were underlined by a faint red fluorescence (Fig. 4b). Mucin was mainly localized in areas rich in cholesterol crystals and seemed to be excluded from the pigment precipitate content; however, large pigment deposits were often covered with a layer of mucin. Using protocol 2, it was possible in some areas to identify clusters of vesicles (Fig. 5a) which were clearly associated with mucin (Fig. 5b). APF was clearly associated with the pigment precipitates and the specific fluorescence was stronger at the periphery of these precipitates (Fig. 6a & Fig. 7).
Fig. 2. Radiated structures, similar to the central core of mixed gallstones, containing pigment and cholesterol crystals (bar I cm=125 pm).
Fig. 3. Crystalline structures, similar to the luminuted periphery of gallstones (bar 1 cm= I25 pm).
354
Immunofluorescence studies of biliary sludge
Fig. 4. Immunolocalization of gallbladder mucin in a sediment of gallbladder bile after ultracentrtfiigation. a) Normal light: cholesterol crystals: plates (*) and tubules (arrowheads), are associated with a granular matrix. b) Fluorescent light: Mucin is located in the granular matrix (A4), only a few cholesterol crystals (arrowheads) are underlined by the Texas-Redjuorescence (bar I cm=125 ,um).
Cholesterol crystals and the matrix were never labeled (Fig. 6b). The aminopeptidase-N was also associated with the pigment precipitates (Fig. 8) and showed the same localization as APE Albumin was scarcely detected in the sediments, but
Fig. 5. a) Lipid vesicle aggregates (arrowheads) in a sediment of gallbladder bile after ultracentrtfugation. b) Lipid vesicles aggregates are clearly labeled by the Texas-Red fluorescence for gallbladder mucin (bar I cm=125 ,um).
if present albumin appeared as little spots which were randomly located. No specific localization was detected for IgA, except in the structures, which looked like the core of gallstones; in this case IgA seemed to be associated with the crystalline part of these structures. 35.5
I? Lechene de la Porte et al. TABLE
1
Concentration difference (meantSEM) of cholesterol, mucin. protein and bilirubin between native and ultracentrifuged gallbladder bile of 49 patients with cholesterol and 16 patients with mixed stones. The percentage of the major components of biliary “sludge” in gallbladder bile is shown in parentheses Cholesterol
Cholesterol Mucin Protein Bilirubin Total sludge quantity
stones
Mixed stones
mg/ml
0%
mg/ml
‘%I
2.08-cO.39 0.41 to.20 1.70t0.51 0.07~0.01 4.2620.78
(48.924.6) (9.623.0) (4O.Oi7.2) (2.711.1)
1.11t0,23 0.15ro.08 1.08t0.20 0.1610.08 2.51 -to.39
(44.427.1) (8.4t3.2) (43.5f4.9) (6.5+1.8)
Quantification of biliary sludge As a result of cholesterol analysis in the collected gallstones, 49 patients (38 women and 11 men) were classified as cholesterol stone (>50’%) cholesterol) carriers. Sixteen patients (10 women and 6 men) had intermediate results (lo--50% cholesterol) and represented the mixed stone group. The major components of biliary sludge, i.e. cholesterol, protein, mucin and bilirubin, were determined as the concentration difference (mg/ml) between native and ultracentrifuged gallbladder bile. The results for the two stone groups are given in Table 1. The findings demonstrated no significant differences in the total amount or the single components of biliary sludge between the two groups of patients. This was due to a wide variation in amounts of sludge in gallbladder bile samples as determined by the ultracentrifugation technique. The calculation derives partially from basal values in native and ultracentrifuged gallbladder bile which have been presented previously (46). The percentages of cholesterol in biliary sludge in the two groups of patients were similar to the percentage of cholesterol in cholesterol or mixed gallstones. These findings and the immunofluorescence studies suggest that biliary “sludge” is a necessary intermediate stage of gallstone formation in these patients.
Discussion Fig. 6. Immunolocalization of APF in a sediment ofgullbladder bile after ultracentrifugution. u) Normal light. Cholesterol crystal (*) und pigment precipitates (P) are embedded in a mucous matrix (M). b) After protocol 2, APF was shown to he associated to the pigment aggregates (P); cholesterol crystals (*) are never labeled by the immunoreaction (bur I cm=125 pm).
356
Gallbladder bile from gallstone patients contains nonsoluble material which comprises a suspension of cholesterol crystals and/or calcium bilirubinate granules embedded in mucin, a mixture of high molecular weight mucus glycoprotein with a low mannose content and proteins (27,28). In the presented study, “sludge” was defined as any non-soluble material in bile which precipitates after 1 h of ultracentrifugation. Instead of centrifugation at
Immunofuorescence
Fig. 7. Irnmunolocalization of APF in a sediment of gallbladder bile after ultracentrifugation after the protocol 1. the specific FITC yellow fluorescence is clearly associated to the periphery of thepigmentprecipitates (*) (bar 1 cm= 500 pm).
3000 g we used ultracentrifugation for 1 h at 100 000 g to separate “sludge” from gallbladder bile to ascertain that any residual cholesterol crystals were sedimented, providing a correct quantification of biliary sludge. In this strict and not generally accepted definition, each bile contains some amount of “sludge”. Due to methodological difficulties in analyzing the components of the sediment after ultracentrifugation an indirect approach was chosen. Measurement of the concentration differences of the main components of sludge between native bile and the supernatant after ultracentrifugation was used. This allowed the determination of comparably tiny amounts of “sludge” not visible in the gallbladder by diagnostic methods like ultrasonography. In addition, the relationship between selected biliary proteins, previously shown to be present in cholesterolcontaining gallstones, and cholesterol crystals or pigments were studied using immunofluorescence. As expected, the morphological studies showed that the pellets were rich in cholesterol crystals at different steps of their formation (47): tubules and the more
studies
of biliary sludge
Fig. 8. Immunolocalization of aminopeptidase N ment of gallbladder bile after ultracentrtfugation protocol I: the specific FITC-yellow fluorescence associated to the pigment precipitates (*) in the as for APF (compare to Fig. 7) (bar I cm=500
in a sediafter the is clearly same way pm).
stable monohydrate cholesterol plates. In all samples studied they were embedded in an uncolored mucous matrix, which also contained small granular structures. Beside these cholesterol crystals, brown pigment precipitates were more or less developed, which also were trapped in the mucous compound. Cholesterol crystals and pigment precipitates seemed to be randomly placed in the gel without any specific relationships; they only co-existed in the same medium. In some cases, structures resembling the radiated central core described in mixed gallstones were present, and cholesterol crystals and pigments were closely associated (Fig. 2). Other elaborate structures (Fig. 3) looked like the periphery of the more developed stones where pigment and cholesterol deposits alternated in concentric areas of mixed stones. These latter structures were rich in cholesterol crystals. Immunolocalization showed that the mucous granule-rich matrix contained mucin, and that cholesterol crystals were clearly associated with it. It is interesting that gallbladder mucin was also clearly associated with clusters of vesicles (Fig. 5) in such a quantity that it was detectable. Using three separate physical chemical 357
P. Lechene de la Porte et al
techniques including dynamic light scattering, transmission electronic microscopy and fluorescent biochemical assays, mucin-vesicle interactions were demonstrated already in model bile (48). Our study in native bile supports this finding that mucin induces vesicle aggregation and fusion and, by this mechanism facilitates cholesterol crystallization in bile. In cholesterol gallstones a very bright mucin signal has been described associated with cholesterol crystals but not with pigment-rich areas (13). Mucin was interlaced among the cholesterol crystals and concentrated at the boundaries between the cholesterol and pigment zones. As in stones, in sludge samples mucin surrounded both the pigmented and cholesterol zones but was never identified within the pigment deposit layers. The concept that mucin gel promotes the crystallization of cholesterol in bile was also supported by a recent study (49). The gel matrix can often become trapped within the growing crystals or between crystals and in mixed gallstones cholesterol crystals appeared to be agglomerated by a thin film of mucin (13). Mucin is also a scaffold on which smaller acidic proteins such as CBP and/or APF are bound, which in turn regulate the deposition of mineral components. In . mixed gallstones (13) APF was demonstrated only in zones which contained calcium salts and bile pigments. It was especially located at the interfaces between cholesterol and pigments deposits. In the sediments investigated we obtained the same kind of localization for APE This small polypeptide (7 kD) is highly amphipathic, leading to avid self-aggregation. It is strong phospholipid and pigment binding as well as bile salts binding (50). In model biles, APF was shown to have an inhibitory function on cholesterol crystallization by stabilizing the vesicles against the effect of calcium (51). In vitro studies with model bile revealed that APF retains cholesterol within supersaturated multivesicular aggregates and markedly decreased the rate of crystal formation when compared to the vesicles without APF (50). When calcium was present in the model bile APF counterbalanced the destabilizing effect of calcium. The crystal mass was decreased in the presence of APF but the nucleation time was not modified. Calcium has been shown to be present within cholesterol stones in several specific areas (51). The concentration of calcium in the center of stones suggested the possibility that calcium salts may act as a nidus for subsequent cholesterol crystallization and stone formation. in the presence of calcium, CBP presented a clear cooperative effect with mucin to promote and aggregate cholesterol crystals (20). In sediments as in stones, a nucleating promotor
358
protein, aminopeptidase-N, was clearly associated with pigment deposits, at their junction to cholesterol at the same localization as APE Albumin was not detected in the “sludge samples” except in some cases as infrequent “spots” randomly located. Similarly, IgA, a protein widely thought to promote cholesterol crystallization showed no specific localization in the sediments or in gallstones (13). Only in a few cases was this protein clearly associated with aggregations of cholesterol crystals which resemble the concentric cholesterol layers at the periphery of stones. Interestingly, biliary secretory IgA has recently been described as a major constituent of a group of cholesterol crystal-binding proteins (53). Thus crystal-binding IgA may be an important modulator of cholesterol crystal agglomeration into stones and stone growth in viva, as supposed by Busch et al. (53). In conclusion, the striking similarities concerning the relationship between cholesterol, biliary pigments and biliary proteins in cholesterol-containing gallstones and in different solid components of the sediment in gallbladder bile suggest strongly that biliary “sludge” is a necessary intermediate step in gallstone formation.
Acknowledgements This paper is dedicated to Gustav Paumgartner for generous support and critical discussion of the subject. Parts of these results were presented at the Annual Meeting of the American Gastroenterological Association in New Orleans, May 1998 and appeared as an abstract in Gastroenterology 1998; 114: A 528.
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