1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Previously, we identified needle-like and filamentous, putatively “anhydrous” cholesterol crystallization in vitro at very low phospholipid concentrations in model and native biles. Our aim now was to address whether spontaneous gallstone formation occurs in Mdr2 (Abcb4) knockout mice that are characterized by phospholipid-deficient bile. Biliary phenotypes and cholesterol crystallization sequences in fresh gallbladder biles and non-fixed liver sections were determined by direct and polarizing light microscopy. The physical chemical nature and composition of crystals and stones were determined by sucrose density centrifugation and before mass and infrared spectroscopy. Gallbladder biles of Mdr2−/− mice precipitate needle-like cholesterol crystals at 12 weeks of age on chow. After 15 weeks, more than 50% of Mdr2−/− mice develop gallbladder stones, with female mice displaying a markedly higher gallstone-susceptibility. Although gallbladder biles of Mdr2−/− mice contain only traces (≤ 1.1 mM) of phospholipid and cholesterol, they become supersaturated with cholesterol and plot in the left 2-phase zone of the ternary phase diagram, consistent with “anhydrous” cholesterol crystallization. Furthermore, more than 40% of adult female Mdr2−/− mice show intra- and extrahepatic bile duct stones. In conclusion, spontaneous gallstone formation is a new consistent feature of the Mdr2−/− phenotype. The Mdr2−/− mouse is therefore a model for low phospholipid-associated cholelithiasis recently described in humans with a dysfunctional mutation in the orthologous ABCB4 gene. The mouse model supports the concept that this gene is a monogenic risk factor for cholesterol gallstones and a target for novel therapeutic strategies. (HEPATOLOGY 2004;39:117–128.)

The multidrug resistance gene 2 (Mdr2) encodes the hepatic canalicular transporter for the major biliary phospholipid phosphatidylcholine (lecithin). This protein belongs to the ATP-binding cassette (ABC) transporter family, and accordingly the official gene symbol Abcb4 has been assigned. Mice with homozygous disruption of the Abcb4 gene (commonly denoted Mdr2−/−- mice) display almost complete absence of phospholipids from bile.1 The phospholipid-deficiency of Mdr2−/− mice2, 3 results in liver injury from chronic cholangitis, resembling human liver disease due to mutations of the orthologous gene ABCB4.4ABCB4 mutations result in a wide spectrum of phenotypes, ranging from progressive familial intrahepatic cholestasis (PFIC type 3) to adult cholestatic liver disorders characterized by elevated γ-GT levels.4 Under healthy conditions, mixed cholesterol-lecithin-bile salt micelles protect the biliary epithelium against the detergent properties of bile salts. The cholestatic liver injury in these patients is therefore commonly attributed to the “toxic” effects of bile salts on the apical membranes of hepato- and cholangiocytes.5 The liver injury in Mdr2−/− mice is characterized by segmental biliary strictures due to periductal fibrosis and fibro-obliteration of bile ducts.6

Biliary lecithin also plays a key role in solubilizing excess cholesterol in the form of unilamellar cholesterol-lecithin-vesicles. In agreement with this paradigm, Rosmorduc et al.7, 8 recently described a clinical entity characterized mainly by the occurrence of intrahepatic and gallbladder microlithiasis in young adults associated with ABCB4 mutations. This “peculiar” form of cholelithiasis was termed low phospholipid-associated cholelithiasis (LPAC). In earlier work, we had identified needle-like and filamentous cholesterol crystallization, apparently “anhydrous” in in vitro studies of model biles9, 10 and native biles with very low phospholipid/bile salt ratios.11, 12 Therefore, we hypothesized here that similar crystallization phenomena would be observed in phospholipid-deficient bile of Mdr2−/− mice in vivo and lead to gallstones. We compared the physical-chemistry of fresh hepatic and gallbladder biles between Mdr2−/− and wild-type mice. Because spontaneous gallstone formation, without a dietary challenge, had not been reported in previous studies of this animal model,1–3, 13–16 we carried out a systematic microscopic as well as physical-chemical analysis of the biliary phenotypes, identifying gallstone formation as an important feature of Mdr2−/− mice.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Chemicals and Standards

For high performance liquid chromatography (HPLC), bile salt standards were purchased from Sigma Chemical (St. Louis, MO) and CalBiochem-Behring (San Diego, CA), with the exception of tauro-β-muricholate and tauro-ω-muricholate, which were generous gifts from Tokyo Tanabe (Tokyo, Japan). The purities of individual bile salts were at least 98% by thin layer chromatography (TLC) or HPLC. Dilauroyl-phosphatidylcholine [PC-(24:0)], dimyristoyl-PC [PC-(28:0)], diarachidoyl-PC [PC-(40:0)], and dibehenoyl-PC [PC-(44:0)] standards for mass spectrometry were obtained from Sigma Chemical with purities of at least 99% by TLC. N-oleoyl-D-sphingomyelin (18:1) was 95% pure by TLC (Sigma Chemical). HPLC reagents and all other chemicals used were of the highest purity commercially available and purchased from Sigma or Fisher Scientific (Pittsburgh, PA).

Gene-Targeted Mice and Diet

Breeding pairs of homozygous Mdr2−/− gene-targeted mice (official name FVB/N-Abcb4tm1Bor) and Mdr2+/+ wild-type mice on the FVB/NJ background were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were housed as groups of 5 per cage in a temperature-controlled room (22 ± 1°C) that was illuminated from 6.00 AM to 6.00 PM.

Genotypes of Mdr2−/− mice were confirmed by polymerase chain reaction (PCR) using neomycin specific primers (5′-CTT GGG TGG AGA GGC TAT TC-3′; 5′-AGG TGA GAT GAC AGG AGA TC-3′) and 10 to 20 ng genomic DNA isolated from individual tail snips by the rapid lysis technique (DNeasy Tissue Kit, Qiagen AG, Hilden, Germany). The PCR reactions contained 2 mM MgCl2, 10 mM dNTPs, 200 nM primers, and 1 U Taq DNA polymerase (Roche Diagnostics, Mannheim, Germany) in 50 μL. PCR cycling conditions were 95°C/30 s, 55°C/60 s, and 72°C/30 s for 35 cycles, and a final extension step of 10 min at 72°C.

The mice were weaned at 3 weeks of age and maintained on laboratory chow diet (Purina 5001, St. Louis, MO) that contains less than 0.02% (wt/wt) cholesterol, as determined by HPLC.17 After 6, 9, 12, and 15 weeks on chow diet (i.e., at 9, 12, 15, and 18 weeks of age, respectively), mice were anesthetized (10 mg Avertin IP, Sigma-Aldrich), and laparotomy and cholecystectomy were performed at 9.00 AM after overnight fasting.12 Gallbladder volumes were determined gravimetrically, assuming a density of 1 g/ml bile.12 In a second group of mice, the lower end of the common bile duct was ligated, the common bile duct was cannulated with a PE-10 polyethylene catheter (Becton Dickinson, Sparks, MD) below the entrance of the cystic duct, which was doubly ligated, and hepatic bile was collected by gravity.18

For routine biochemistry, serum samples were stored at −70°C until analysis of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), and bilirubin by routine clinical chemistry testing on a Hitachi 717 analyzer (Boehringer Mannheim, Mannheim, Germany).

Mice received care according to the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences (NIH publication 86-23). Protocols were approved by the Animal Care and Use Committees of Harvard University and Aachen University. Mice were monitored routinely for selected infectious agents, and gallbladder bile samples were cultured to exclude bacterial infection. As expected for rodents, all mice showed progressive weight gain from 20 to 26 gm at 9 weeks of age to 24 to 30 gm by the end of the experiments.1, 2

Microscopic Studies

For detection of cholesterol crystals and quantification of gallstone number and size, the gallbladder of each mouse was opened at the fundus. The observers (D.Q.-H.W., F.L.) were unaware of the animals' genotypes. Fresh gallbladder biles were then observed using direct and polarizing light microscopy (Leitz microscope DM RB, Leica Microsystems, Wetzlar, Germany) without a cover slip.10, 12 After compression with a cover glass, phase contrast and Nomarski differential interference optics were also employed. Liquid crystals and solid cholesterol crystals were defined according to previous criteria.10–12 Also, 5 μL of fresh hepatic biles were examined microscopically (n = 5 per group). In addition, bile samples were incubated at 37°C under argon for up to 7 days and followed by sequential microscopic examination.

For liver histology, tissue specimens were fixed in 10% formalin, and paraffin sections were stained with hematoxylin and eosin (HE), or 0.1% Sirius red F3B in saturated picric acid (Chroma, Münster, Germany).19 For visualization of intrahepatic cholesterol crystals, tissue was cryostat-sectioned at 5-μm thickness, rinsed in water, and mounted. Crystals were visualized by polarizing light microscopy.

Infrared Spectroscopy

Gallstone composition was analyzed by infrared spectroscopy, as described previously.20, 21 Gallstones were finely ground with an agate mortar and pestle, and compressed with spectral grade KBr at 8 t/cm2 to form thin wafers. Infrared spectra between 4,000 and 400 cm-1 were recorded for 8 to 12 min and evaluated manually and by computer-assisted comparison with previously recorded spectra.20, 22

Sucrose Density Gradient Centrifugation

Cholesterol crystals were concentrated by ultracentrifugation using a tabletop ultracentrifuge at room temperature, layered on top of linear 2 to 20% sucrose density gradients, and centrifuged for 1 h at 201,000 g.23, 24 Fractions were aspirated from top to bottom and examined by polarizing light microscopy, as described above. Sucrose densities were verified using a temperature-compensated refractometer.

Cholesterol monohydrate and anhydrous cholesterol crystals were used as standards. To prepare cholesterol monohydrate crystals,25 cholesterol (Nu-Chek Prep Inc., Elysian, MN) was recrystallized 3 times from hot 95% (vol/vol) ethanol. The final solution was cooled to room temperature, and the cholesterol monohydrate crystals were harvested by filtration, washed repeatedly with water, and dried overnight in a dessicator thermostated at 40°C. For anhydrous cholesterol crystals,25 cholesterol was dissolved in hot (70°C) glacial acetic acid and the solution was allowed to cool. The crystals were harvested as described above and placed in a heated dessicator (80°C) overnight under vacuum to remove all remaining acetic acid.

Biliary Lipid Analysis

Lipids were extracted according to Bligh and Dyer.26 Total biliary phospholipid concentrations were measured as inorganic phosphorus by the method of Bartlett27 as well as enzymatically using phospholipase D and choline oxidase,28 employing an assay kit from Wako Chemicals GmbH (Neuss, Germany). Cholesterol and bile salt concentrations were determined using cholesterol oxidase and 3α-hydroxysteroid dehydrogenase, respectively, as described previously.12 Cholesterol saturation indexes (CSIs) of bile samples were calculated from the critical tables.29 Individual bile salts were determined by HPLC.12

Measurement of Biliary Phospholipid Molecular Species

Biliary lipid extracts from 10 μl of gallbladder bile were taken to dryness under N2, redissolved in 20 to 100 μl methanol/chloroform (2:1, vol/vol), and centrifuged in a tabletop centrifuge at 16,000 rpm for 5 min. Samples were processed exclusively in glassware to avoid contamination with plasticizers and related materials. Mass spectrometric analyses were performed with the triple quadrupole instrument TSQ 7000 (Finnigan MAT, Bremen, Germany) equipped with a nanoelectrospray source, as described previously.30, 31 After single-stage positive ion scans, PC and sphingomyelin molecular species were selectively analyzed in the tandem mode by (i) scanning for neutral loss of the choline phosphate head group (183 Dalton) from cationized PC or sphingomyelin molecules ([M+Na]+ adducts) and (ii) precursor scanning at m/z 184, which detects [M+H]+ ions of choline-containing phospholipids only.31 For each spectrum, 50 to 150 repetitive scans were averaged. To correct for the influence of the fatty acid chain length on the signal intensity of PC, a set of standards [PC-(24:0), PC-(28:0), PC-(40:0), PC-(44:0)] was used for calibration according to Brügger et al.31

Statistical Methods

Data are expressed as means ± SE. Four to 8 animals from each group were studied at each time point. For evaluation of statistical significance, differences were assessed by Student's t-test for independent samples. Statistical significance was defined as a 2-tailed probability P less than .05 unless otherwise stated. Analyses were performed with SPSS 10 software (SPSS, Chicago, IL).


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Gallbladder Bile of Mdr2−/− Mice Precipitates Unusual Solid Crystals

Upon laparotomy, careful macroscopic and microscopic examination of the biliary system of female Mdr2−/− mice at 12 weeks of age and males at 15 weeks of age revealed the presence of needle-shaped crystals and stones in the gallbladder. No crystals or stones were observed before this age. Of note is that the bile of Mdr2−/− mice did not contain helical or tubular cholesterol crystals,10–12 and cholesterol monohydrate crystals, which are characterized by a plate-like shape and often display a notched corner, were not observed either.25 Figure 1 shows photomicrographs of typical gallstones. Individual needle-like crystals project from the edges of the stones, which also contain amorphous mucin gel (Fig. 1A). When the stones were broken by a slide cover, we found the core of stones to be composed of needle-like crystals and gelled mucin (Fig. 1B). The needle-like crystals are short, straight filamentous crystals (<20 μm). Figure 2 displays representative microscopic images of these crystals with Nomarski differential interference optics, indicating that their morphology resembles the filamentous and needle-like cholesterol crystals described previously by our group in model biles of similar low phospholipid composition.9, 10

thumbnail image

Figure 1. Photomicrographs of gallbladder stones observed in Mdr2−/− mice. Figures were obtained using the Leitz microscope DM RB (magnification 400×) at room temperature (20°C). To highlight the birefringence of the solid crystals, the microscope was operated with crossed polars in operation without a first order quartz compensator. (A) Needle-like crystals (arrows) project from the edges of the yellowish stone, which also contains amorphous material. The needle-like crystals are short, straight, filamentous cholesterol crystals (<20 μm). The black background indicates the absence of liquid crystals. (B) Radial crystal pattern of a stone's core composed of needle-like crystals (arrow) and gelled mucin.

Download figure to PowerPoint

thumbnail image

Figure 2. Photomicrographs of crystals observed in gallbladder bile of Mdr2−/− mice, using Nomarski differential interference optics. The bile contains multiple freely floating, short, needle-like crystals (A), as well as longer, filamentous, and arc-like crystals (B). Furthermore, agglomerated crystals, often surrounded by mucin gel, are observed. Magnification 400×.

Download figure to PowerPoint

Time Course and Gender Difference of Cholesterol Crystallization and Gallstone Formation

Figure 3 shows the prevalence of needle-like crystals and stones as functions of age for female (left panel) and male (right panel) Mdr2−/− mice. At 9 weeks of age, the macroscopic and microscopic examination of gallbladder biles shows no evidence of crystals or gallstones. However, mucin gel is detected in 20% of female Mdr2−/− mice. At 12 weeks, gallbladder biles of female Mdr2−/− mice contain needle-like crystals (33%) as well as gallstones (50%), and stone prevalence increases strongly with passage of time. At 15 weeks, 60% of female Mdr2−/− mice display needle-like crystals and 80% show gallstones. By 18 weeks, all female mice develop stones (Fig. 3, left panel). In male Mdr2−/− mice, needle-like crystals and gallstones form later (≥ 15 weeks) and at lower prevalence rates (Fig. 3, right panel) compared to female mice.

thumbnail image

Figure 3. Percent of female Mdr2−/− mice (left panel) and male Mdr2−/− mice (right panel) forming free needle-like crystals and gallbladder stones as functions of age in weeks. Open squares (□) represent crystals; closed circles (•) stones. Crystal and stone prevalences increase with age. In male mice, needle-like crystals and stones appear later and at lower prevalence rates compared to female mice.

Download figure to PowerPoint

As expected, gallbladder biles of Mdr2+/+ (wild-type) mice are microscopically free of crystals or stones. In addition, we tested heterozygous Mdr2+/− mice at 12 and 15 weeks of age (n = 10–14), whose gallbladder biles do not precipitate crystals either. Furthermore, all gallbladder biles of both Mdr2+/+ and Mdr2+/− mice remain crystal-free when followed by sequential microscopic examination during incubation at 37°C for 7 days.

Number, Size, and Composition of Gallstones

Figure 4 shows the average gallstone numbers and diameters in gallbladders of female and male Mdr2−/− mice. The number of gallstones per mouse ranges from 1 to 8 with an average of 1.3 ± 0.3 stones per mouse. Most gallstones are smaller than 0.1 mm, with a mean stone size of 0.15 ± 0.04 mm. On average, both stone number and size are significantly higher in female compared to male mice.

thumbnail image

Figure 4. Gallstone characteristics in Mdr2−/− mice. Average gallstone numbers per mouse (upper panel) and gallstone diameters (lower panel) are shown as functions of age in weeks. Twelve-week-old female knockout mice, but not male mice present with stones. At 15 weeks of age, gallbladders of female Mdr2−/− mice contain significantly more gallstones compared to male mice; moreover at 18 weeks of age, gallstones in female mice are significantly larger than in males. Double asterisks (**) indicate P less than .01, single asterisks (*) P less than .05, compared to male mice.

Download figure to PowerPoint

Ultracentrifugation of gallbladder bile samples from Mdr2−/− mice on sucrose density gradients yields needle-like crystals at intermediate densities (d = 1.03–1.04 gm/ml) that maintain their crystal habits in water for up to 48 h. Standard needle-like anhydrous cholesterol and plate-like cholesterol monohydrate crystals (see Materials and Methods) result in 2 bands at d = 1.03 and d = 1.05 gm/ml, respectively. This indicates that the crystals harvested from Mdr2−/− mice may be anhydrous cholesterol, which undergoes subsequent hydration as water becomes incorporated within the crystal lattice.10, 23, 32 This phase transition has been confirmed recently by grazing incidence x-ray diffraction analysis of ultrathin cholesterol films on water.33

However, gallbladder stones of Mdr2−/− mice demonstrate densities greater than 1.05, consistent with stones containing additional compounds beside cholesterol, particularly bilirubin with d = 1.31 gm/ml.34 Indeed, trace quantities of unconjugated bilirubin are detected by mass spectroscopy (m/z 584) in gallstones of Mdr2−/− mice. However, infrared spectroscopy reveals pronounced bands at 1,032, 602, and 562 cm−1, demonstrating calcium phosphate to be the most likely major inorganic stone compound (80%). The predominant calcium salt was likely to be carbonate (apatite), as indicated by the typical absorption bands at 1,449, 1,415, and 873 cm−1.20

Biliary Lipid Compositions

Table 1 shows the biliary lipid compositions of gallbladder biles from Mdr2+/+ and Mdr2−/− mice of both genders on chow. As has been shown for other inbred mouse strains,12 the gallbladder biles of wild-type mice are not supersaturated with cholesterol with CSIs less than 1.00. The predominant phospholipid class in wild-type mice is phosphatidylcholine (PC), whereas sphingomyelin (SM) comprises only 2% of total phospholipids (Table 1). As expected for mice with homozygous disruption of the PC transporter Mdr2,3, 16 only minimal amounts of PC (<3% of controls) are detected in biles of Mdr2−/− mice. This decrease is not compensated for by SM, with SM levels being low and essentially identical to those in wild-type mice.

Table 1. Biliary Lipid Compositions of Gallbladder Biles
Mouse StrainaGenderAge (weeks)Ch (mM)PC (mM)bSM (mM)bBS (mM)Ch/PLCh (mole %)PL (mole %)BS (mole %)[TL] (g/dL)CSIc
  • a

    Values were determined in 4–8 animals per group.

  • b

    In Mdr2+/+ mice, PC and SM concentrations were determined by ESI-MS/MS. SM concentrations were calculated based on the 4 major species detected (16:0, 22:0, 24:0, 24:1).

  • c

    For Mdr2+/+ mice, CSI values were calculated using the critical tables,29 and thus are estimates based on taurocholate; for Mdr2−/− mice, calculations were based on the bile salt pool compositions of Mdr2−/− mice (see Table 2) and the maximal cholesterol-solubilizing capacities of micellar solutions of the two principal taurine conjugated bile salts.35, 36

  • **, d, ‡

    *P < 0.05; **P < 0.01, compared to Mdr2+/+ mice; P < 0.05, compared to 9 weeks.

  • e

    In 18-weeks-old male Mdr2+/+ mice, analysis of biliary lipids in the isotropic phase of gallbladder bile was not feasible due to the development of minuscule gallbladders (mean volume 2 ± 1 μl) filled with sticky mucin gel and needle-like crystals (see Results on gallbladder volumes).

  • Abbreviations: BS, bile salts; Ch, cholesterol; CSI, cholesterol saturation index; F, female; M, male; Mdr2, multidrug resistance gene 2; ND, not determined; PC, phosphatidylcholine; PL, phospholipids; SM, sphingomyelin; [TL], total lipid concentration.

Mdr2+/+M12–154.99 ± 0.1819.12 ± 0.290.51 ± 0.13216.3 ±
Mdr2+/+F12–155.80 ± 0.6323.35 ± 1.620.44 ± 0.15256.2 ±
Mdr2+/+M and F12–155.39 ± 0.3521.24 ± 1.390.47 ± 0.09236.3 ±
Mdr2−/−M90.24 ± 0.010.50 ± 0.04ND218.0 ± 15.70.480.110.2399.6611.770.91
Mdr2−/−F90.30 ± 0.010.44 ± 0.03ND214.5 ± 19.90.680.140.2099.6611.580.65
Mdr2−/−M and F90.27 ± 0.010.47 ± 0.03ND216.3 ± 11.80.570.120.2299.6611.680.78
Mdr2−/−M12–150.26 ± 0.060.46 ± 0.130.48 ± 0.19203.3 ± 12.40.570.130.4699.4111.011.08
Mdr2−/−F12–150.46 ± 0.160.30 ± 0.090.19 ± 0.06212.5 ± 17.31.530.220.2399.5611.481.05
Mdr2−/−M and F12–150.36 ± 0.09**d0.37 ± 0.10**0.34 ± 0.11209.7 ± 12.50.970.17**0.34**99.49**11.341.07**
Mdr2−/−Fe181.09 ± 0.64d0.38 ± 0.02ND225.8 ± 13.22.870.480.1799.3512.212.24
Table 2. Bile Salt Molecular Species in Gallbladder Biles
Mouse Strain*GenderT-β-MCT-ω-MCTCTCDCTDCHI
  • *

    Values were determined by HPLC from pooled gallbladder biles (n = 10) and are given in percent.

  • The hydrophobicity indexes for bile salts are calculated according to Heuman.38

  • Abbreviations: F, female; HI, hydrophobicity index; M, male; ND, not detectable; Mdr2, multidrug resistance gene 2; TC, taurocholate; TCDC, taurochenodeoxycholate; TDC, taurodeoxycholate; T-β-MC, tauro-β-muricholate; T-ω-MC, tauro-ω-muricholate.

Mdr2+/+M and F57.−0.45
Mdr2−/−M and F49.2ND48.50.30.7−0.38

Although biles of Mdr2−/− mice contain traces only of cholesterol (Table 1), they are supersaturated with cholesterol (CSI ≥1.05) in 12- to 15-week-old mice, when calculated for the maximal cholesterol-solubilizing capacities of micellar bile salt solutions in the virtual absence of PC.35, 36 Because different species and compositions of bile salts could influence cholesterol solubility, we studied model biles of cholesterol-lecithin-taurocholate-tauro-β-muricholate, which generated 2 new phase diagrams with a mixture of taurocholate-tauro-β-muricholate in a ratio of 1:1 (wt/wt) according to the analyzed bile salt species (Table 2) and the average total lipid concentrations (∼10 and 15 gm/dl) of the pooled gallbladder biles of Mdr2+/+ (Fig. 5, top panel) and Mdr2−/− mice (bottom panel).10, 12, 37 For purposes of illustration, we plot in Fig. 5 the relative biliary lipid compositions of Mdr2+/+ mice (top panel) and Mdr2−/− mice (bottom panel) on condensed ternary cholesterol-lecithin-taurocholate-tauro-β-muricholate phase diagrams. Also, we studied mixtures down to ∼0.4% phospholipids, and defined phase boundaries for biles with very low phospholipid/bile salt ratios (see the inset of Fig. 5). Gallbladder biles of Mdr2+/+ mice plot in the micellar zone, that is, that they are not saturated with cholesterol (CSI ≤0.53) and do not precipitate crystals. The cholesterol supersaturated biles of Mdr2−/− mice plot in region A of the left 2-phase zone. By phase analysis in model bile systems,10 biles in region A are predicted to contain needle-like, putatively “anhydrous” cholesterol crystals, exactly as observed in our experimental analysis (Figs. 2 and 3). These findings explain physical-chemically why Mdr2−/−- mice develop gallstones composed of an unusual crystalline cholesterol habit. Furthermore, during the experimental time course, biliary cholesterol concentrations and CSIs increase and PC concentrations decrease markedly (Table 1), consistent with onset of stone formation after 12 weeks of age.

thumbnail image

Figure 5. Relative biliary lipid compositions of gallbladder biles of Mdr2+/+ control mice (upper panel) and Mdr2−/− knockout mice (lower panel) and plotted as mole% on condensed trilinear phase diagrams. The phase boundaries are drawn according to the mean total lipid concentrations (TL) and compositions of the bile samples (data from Tables 1 and 2).10, 12, 37 The solid curved line encloses the one-phase micellar zone. Above the micellar zone, 2 solid and 2 dashed lines divide the phase diagrams into regions A to E with different crystallization sequences (see Wang and Carey10). The area of detail depicts the lower left corner of the lower phase diagram for biles with very low phospholipid/bile salt ratios. Closed symbols (•) represent lipid compositions of male mice; corresponding open circles (○) compositions of female mice at 12–15 weeks of age. Gallbladder biles of Mdr2+/+ mice plot in the micellar zone; biles of Mdr2−/− mice plot in region A the lower left corner of the phase diagram. These biles are characterized by very low phospholipid/bile salt ratios, cholesterol (super)saturation, and precipitation of needle-like cholesterol crystals.

Download figure to PowerPoint

Table 2 summarizes the relative bile salt compositions of pooled gallbladder biles (n = 10) of Mdr2−/− and wild-type mice. Tauro-β-muricholate and taurocholate are the predominant bile salts, comprising more than 97% of the bile salt pool. Of note, gallbladder bile of female Mdr2−/− mice contains higher taurocholate and lower tauro-β-muricholate levels than bile of male knockout mice, which is reflected by a markedly higher hydrophobicity index (HI) of the bile salt pool (−0.24 vs. −0.51). For both genders, bile salt compositions do not differ between wild-type and knockout mice.

Table 3 tabulates the molecular species of PC in gallbladder biles of Mdr2−/− and wild-type mice, as determined by electrospray ionization tandem mass spectrometry (ESI-MS/MS). For the major biliary PC species, no marked sex differences are observed. Three major PC species are present in wild-type mice: 16:0 to 18:2 (51.4%), 16:0 to 18:1 (19.7%), and 16:0 to 20:4 (∼10%). In addition, minor amounts of a variety of other PC species are detected. In contrast to Mdr2+/+ mice, bile of Mdr2−/− mice contains trace amounts of all PC species. Compared to wild-type mice, the relative compositions shift to greater proportions of the more hydrophobic PC-(18:0–18:2) and PC-(18:0–20:4) in knockout mice, which is reflected by a higher PC hydrophobicity index (0.36 vs. 0.23).

Table 3. Phosphatidylcholine Molecular Species in Gallbladder Biles
Mouse StrainaGender32:1b 16:0–16:134:2 16:0–18:234:1 16:0–18:136:4 16:0–20:4 (18:2–18:2)36:2 18:0–18:2 (18:1–18:1)38:6 18:2–20:4 (16:0–20:6)38:4 18:0–20:4 (18:1–20:3)HIc
  • a

    Values were determined by ESI-MS/MS from gallbladder biles (n = 4) and are given in mean percent.

  • b

    Total fatty acid carbon number:number of double bonds. The fatty acid composition of the main phosphatidylcholine molecular species in murine bile30 is given below each additive value.

  • c

    The hydrophobicity indexes for phosphatidylcholines are calculated according to Hay et al.39

  • *, d, **

    *P < 0.01; **P < 0.05, compared to control mice.

  • Abbreviations: F, female; HI, hydrophobicity index; M, male; Mdr2, multidrug resistance gene 2.

Mdr2+/+M and F0.951.419.712.
Mdr2−/−M and F5.217.8*d13.9*21.7*14.6**9.6*17.20.36**

Gallbladder Volumes

The gallbladder volumes of Mdr2+/+ mice range from 2 μl to 10 μl and are similar to values obtained for other inbred strains of mice.12, 17 At 9 weeks of age, gallbladders of male and female Mdr2−/− mice are 3 times larger than gallbladders of wild-type mice (9 ± 1 μl vs. 3 ± 1 μl, P <.01). With passage of time, gallbladder sizes decrease significantly (P <.01) in male Mdr2−/− mice from 11 ± 1 μl at 9 weeks to 2 ± 1 μl at 18 weeks of age, with several old male mice showing very small gallbladders filled with sticky mucin gel and needle-like crystals. Gallbladder sizes of female Mdr2−/− mice (12 ± 1 μl) are significantly (P <.01) larger compared to male mice and do not decrease with age.

Mature Mdr2−/− Mice Develop Bile Duct and Intrahepatic Stones

Figure 6 illustrates that Mdr2−/− mice of both sexes display enlarged, but otherwise normal livers. However, as illustrated in Fig. 7, histologic assessment of liver injury reveals characteristic changes. Whereas parenchymal damage is mild, Mdr2−/− mice display marked proliferation of bile ducts, which is accompanied by periportal and periductal fibrosis, resulting in fibro-obliteration and segmental strictures of bile ducts (Fig. 7D and E). Table 4 shows that the cholestatic liver damage in Mdr2−/− mice is reflected by significantly elevated serum transaminase and AP activities, as well as slightly increased bilirubin levels, similar to previous reports.1, 2, 6 These findings demonstrate that the liver injury of Mdr2−/− mice resembles human diseases characterized by progressive sclerosing cholangitis,6 for example, primary sclerosing cholangitis and PFIC (type 3), the latter being caused by ABCB4 deficiency and the resulting absence of biliary phospholipids.4

thumbnail image

Figure 6. Macroscopic appearance of livers from 9- and 18-week-old female (F) and male (M) Mdr2−/− mice in comparison to livers from Mdr2+/+ controls. The livers of knockout mice are enlarged compared to wild-type mice, but look otherwise normal. Liver weights and liver/body weight ratios of Mdr2−/− mice are significantly (P <.01) larger compared to wild-type controls, but do not increase within the experimental period. At 9 weeks, liver weights were 2.4 ± 0.1 g in male and 2.0 ± 0.1 g in female knockout mice, corresponding to liver/body weight ratios of 6.5 and 7.1%, respectively. In contrast, livers of male and female control mice weighed 1.5 ± 0.1 g (liver/body weight ratio 4.9%) and 1.2 ± 0.1 g (4.5%).

Download figure to PowerPoint

thumbnail image

Figure 7. Nonsuppurative inflammatory cholangitis, periductal fibrosis, and hepatolithiasis in Mdr2−/− mice. Light microsopic overviews (HE staining) of the livers of 9-week-old (A) and 18-week-old (B) female Mdr2−/− mice demonstrate proliferation of small bile ducts extending into the periphery. Larger bile ducts of a 9-week-old Mdr2−/− mouse show pronounced periductal fibrosis (D, arrow heads) (6), and an 18-week-old female Mdr2−/− mouse displays progressive ductular proliferation, with multiple ductular cross-sections (stars) indicating serpentine bile ducts (E) (HE staining). Similar but less pronounced histologic changes are observed in male Mdr2−/− mice (data not shown). Cholangitis in Mdr2−/− mice is accompanied by moderate fibrous expansion of portal areas, as demonstrated by Sirius red staining of collagen fibers (F), whereas Mdr2+/+ controls show normal liver sections without fibrosis (C). Polarizing light microscopy of frozen liver sections of an Mdr2−/− mouse at 10 months of age shows an occluded intrahepatic bile duct, which is filled with needle-like crystals (H). Panel I displays the intrahepatic agglomerated needle-like crystals (arrows) at higher magnification. The corresponding HE stain visualizes the periductal fibrosis (G, arrow heads). Original magnifications: A, B, C, F, 50×; D, E, G, H, 100×; I, 400×. pv = portal vein.

Download figure to PowerPoint

Table 4. Liver Chemistry Tests
Strain Mdr2+/+Mdr2−/−
Age   9 Weeks18 Weeks
  • a

    Values were determined in 3–6 animals per group and represent means ± SE.

  • ‡‡, b, **, ##, ‡, *

    *P < 0.05; **P < 0.01, compared to Mdr2+/+ mice; ‡P < 0.05; ‡‡P < 0.01, compared to male mice; #P < 0.05, ##P < 0.01, compared to 9 weeks.

  • Abbreviations: ALT, alanine aminotransferase; AP, alkaline phosphatase; AST, aspartate aminotransferase; F, female; M, male.

ASTa(U/I)137 ± 1347 ± 8‡‡b140 ± 11197 ± 38**214 ± 18##301 ± 23**,
ALT(U/I)44 ± 527 ± 5601 ± 9**654 ± 8**,‡‡230 ± 14**,##258 ± 17**,##
AP(U/I)148 ± 4138 ± 11381 ± 46**395 ± 55*377 ± 20**601 ± 79**,
Bilirubin(μmol/l)14 ± 26 ± 220 ± 412 ± 38 ± 110 ± 3

Upon macroscopic examination of the biliary system of 7-month-old female Mdr2−/− mice we noted dilated extrahepatic duct, which contain multiple concrements impacted in the ducts. By polarizing light microscopy, these show stones of similar morphology compared to the gallbladder stones (Fig. 1) and are composed of needle-like crystals. Whereas only very few mice display bile duct stones by 18 weeks of age, 42% of 7- to 10-month-old female but no male mice develop choledocholithiasis (n = 26). Panels H and I of Fig. 7 display photomicrographs of frozen liver sections of these mature mice using polarizing light microscopy and reveal occluded intrahepatic bile ducts, which are filled with crystalline occlusions composed of needle-like crystals plus mucin gel. In contrast, fresh hepatic biles collected via an acute biliary fistula do not contain any solid crystals when analyzed by light or electron microscopy (data not shown).


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

A major finding of this study is that spontaneous cholesterol stone formation in gallbladder bile of Mdr2−/− mice in vivo confirms predictions from in vitro studies that studied model biles with very low phospholipid/bile salt ratios.9, 10, 40 Moreover, the findings in the Mdr2−/− mouse model are in agreement with recent observations of ABCB4 mutations in adult patients with cholesterol gallstones.7 Rosmorduc et al.7, 8 showed that ABCB4 loss-of-function mutations in either the homozygous or heterozygous state are linked with LPAC. LPAC is characterized by (i) clustering within families; (ii) onset of symptoms before the age of 40 years; (iii) intrahepatic hyperechoic foci, intrahepatic sludge, or microlithiasis; and (iv) recurrence of symptoms after cholecystectomy.7, 8

The link between Mdr2 deficiency and cholelithiasis is consistent with the increased prevalence of gallstones in PFIC type 34 and intrahepatic cholestasis of pregnancy,41 which are associated with ABCB4 mutations in individual patients.4 Shoda et al.42 demonstrated recently that the formation of cholesterol and cholesterol-rich brown pigment stones in intrahepatic bile ducts may be caused by a decreased hepatic efflux of phospholipids because these patients showed significantly decreased levels of ABCB4 mRNA and protein levels as well as reduced phospholipid concentrations in gallbladder bile.42 Fracchia et al.43 further supported this concept by demonstrating that patients with cholesterol microlithiasis display significantly decreased mean percent molar concentrations of phospholipids in duodenal bile. Interestingly, using polarizing light microscopy of hepatic cryostat sections, we observed intrahepatic crystal agglomerates in 7-month-old Mdr2−/− mice (Fig. 7). It is unlikely that the full picture of fibro-obliterative sclerosing cholangitis observed in 8-week-old Mdr2−/− mice6 is the consequence of stone formation, because no stones are present in male animals around that time and fresh hepatic bile samples do not contain any crystals. Moreover, periductal fibrosis and other features of bile duct damage are seen as soon as 2 weeks after birth.6 We therefore speculate that stagnation of bile as a result of strictures and segmental biliary obstruction may promote intrahepatic stone formation in the presence of an abnormal bile composition. Notwithstanding, this study establishes the Mdr2−/− mouse as an animal model for low phospholipid-associated microlithiasis and primary hepatolithiasis. With respect to low phospholipid secretion rates in human disease, it was reported that some non-obese human gallstone patients display significantly reduced phospholipid secretion rates that were out of proportion to low bile salt secretion rates.44 This suggests that the regulation of the hepatic PC transporter might be defective in some non-obese patients with cholesterol gallstone disease. Recently, needle-like cholesterol crystals have been documented in patients with acalculous gallbladder disease,45 albeit the possible pathophysiologic link of this disease entity to low biliary phospholipid concentrations remains to be investigated.

Whereas recent studies in humans7, 8 identified ABCB4 as monogenetic risk factor for cholelithiasis, the Mdr2−/− mouse provides a physical-chemical explanation for the formation of gallstones. Cholesterol and lecithin molecules are secreted by individual transporters from the canalicular membrane and form vesicles within the canalicular space.46, 47 The compositions of mixed vesicles can be inferred from the cholesterol/lecithin molar ratio in bile. Cholesterol-supersaturated vesicles with a cholesterol/lecithin ratio of ∼0.50 precipitate classic plate-like cholesterol crystals.18, 46 In contrast, biles of Mdr2−/− mice (Table 1) can display a cholesterol/lecithin ratio greater than 1.00 (0.65–2.24). Similar to Mdr2−/− mice, bile compositions of LPAC patients show cholesterol supersaturation in conjunction with low phospholipid concentrations, resulting in cholesterol/lecithin ratios between 0.44 and 0.93.7, 43 The bile compositions of Mdr2−/− mice plot in the lower left region A of the biliary phase diagram, which nucleates filamentous and needle-like cholesterol crystals.10 However, the present study cannot discriminate whether the primary crystalline form is truly “anhydrous” cholesterol that undergoes a polymorphic transition or represents a novel monohydrate polymorph, as indicated by a distinct diffraction pattern upon electron diffraction of single crystals in nucleating model bile of similar lipid composition to that of Mdr2−/− mice (Dr. F.M. Konikoff, Tel Aviv University, Dr. Y. Talmon, Technion University of Haifa, personal communication, June 2003). Cholesterol crystals in Mdr2−/− mice could originate from bile salt-dissolution of residual cholesterol-rich vesicles15; alternatively, as evidenced by their increased hydrophobicity indexes (Table 3), direct bile salt elution of structural phospholipids and cholesterol from the canalicular membrane might occur.

Besides the Mdr2−/− mouse, the deer mouse (Peromyscus maniculatus) is the only animal known to form gallstones spontaneously in the wild,48 whereas all other animal models require dietary challenges to increase cholesterol concentrations in bile. Following this paradigm, Mdr2−/− mice were reported to develop gallbladder stones upon dietary challenge with cholic acid (0.1%) for more than 3 weeks,13, 16 but spontaneous gallstone formation has not been recorded in this knockout mouse model. However, development of gallstones in Mdr2−/− mice resembles stone formation in guinea pigs, which are also characterized by very low biliary phospholipid/bile salt ratios.49 Under specific conditions such as vitamin C deficiency or lincomycin-treatment, gallstones of guinea pigs contain randomly aggregated needle-like cholesterol crystals, as well as high amounts of calcium carbonate and calcium phosphate,50, 51 all of which are reminiscent of stones of Mdr2−/− mice. The carbonate content is likely to result from increased bicarbonate secretion in obstructed bile ducts.52

From our model bile systems, we predicted that with a dearth of phospholipids, a cholesterol/phospholipid ratio of ∼1.0 (Table 1) results in precipitation of short needle-like crystals that remain stable and do not evolve rapidly into typical plate-like cholesterol monohydrate crystals.53 In fact, this crystal type predominates in bile of Mdr2−/− mice. The bile of Mdr2−/− mice contains trace amounts of phospholipids, whose molecular species may influence cholesterol crystal habits and transition sequences of crystal intermediates during cholesterol crystallization from bile salt-rich biles.53 Whereas common biliary PC species (16:0–18:2, 16:0–18:1) are detected in bile of Mdr2+/+ mice, bile of Mdr2−/− mice contains rarer structural membrane PCs that presumably are not pumped out by MDR2 (Table 3). The presence of these and other membrane lipids (data not shown) can be attributed to bile-salt induced biliary mucosal injury in Mdr2−/− mice.5 Compared to controls, the bile is relatively depleted in PC species with saturated acyl chains and contains higher amounts of long unsaturated PCs. In vitro, these more hydrophobic PC species are associated with retarded precipitation of short filaments and no formation of metastable helical or tubular intermediates32, 54; and in fact, no intermediate helix formation9, 10 was observed in gallbladder biles of Mdr2−/− mice in vivo.

In addition to phospholipids, bile salt molecular species can influence cholesterol crystallization. In this study, female Mdr2−/− mice display a more hydrophobic bile salt pool due to lower tauro-β-muricholate levels in bile (Table 2). Increasing bile salt hydrophobicity has been demonstrated in vitro to enhance cholesterol crystallization by reducing the liquid crystal region E of the phase diagram, where solid crystal precipitation is a forbidden phase transition.10, 40, 55 This likely explains the earlier onset and higher prevalence of gallstones in female Mdr2−/− mice. These findings provide a rationale for substitution of more hydrophobic bile salts with the more hydrophilic ursodeoxycholic acid in LPAC patients. Moreover, the Mdr2−/− model predicts that a more hydrophilic bile salt pool might not be sufficient to fully prevent cholecysto- and hepatolithiasis associated with phospholipid deficiency and might even display deleterious effects in the presence of fully developed strictures and mechanical obstruction.6 In the future, the Mdr2−/− mouse is expected to serve as an experimental model for the design and testing of new therapeutic interventions for human low phospholipid-associated cholelithiasis.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Dr. Wang is a recipient of a New Scholar Award from the Ellison Medical Foundation (1999–2003). The authors are indebted to Prof. Dr. Wolf D. Lehmann (Central Spectroscopy Department, German Cancer Research Center, Heidelberg, Germany) who generously provided tandem mass spectrometry for analysis of biliary phospholipids and to Dr. Reinhart Kluge (Aachen University) for veterinarian support.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Smit JJM, Schinkel AH, Oude Elferink RPJ, Groen AK, Wagenaar E, van Deemter L, Mol CAA, et al. Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell 1993; 75: 451462.
  • 2
    Mauad TH, van Nieuwkerk CMJ, Dingemans KP, Smit JJM, Schinkel AH, Notenboom RGE, van den Bergh Weerman MA, et al. Mice with homozygous disruption of the mdr2 P-glycoprotein gene. A novel animal model for studies of nonsuppurative inflammatory cholangitis and hepatocarcinogenesis. Am J Pathol 1994; 145: 12371245.
  • 3
    Oude Elferink RPJ, Ottenhoff R, van Wijland M, Smit JJM, Schinkel AH, Groen AK. Regulation of biliary lipid secretion by mdr2 P-glycoprotein in the mouse. J Clin Invest 1995; 95: 3138.
  • 4
    Jacquemin E, de Vree JM, Cresteil D, Sokal EM, Sturm E, Dumont M, Scheffer GL, et al. The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis of adulthood. Gastroenterology 2001; 120: 14481458.
  • 5
    Puglielli L, Amigo L, Arrese M, Nunez L, Rigotti A, Garrido J, Gonzalez S, et al. Protective role of biliary cholesterol and phospholipid lamellae against bile acid-induced cell damage. Gastroenterology 1994; 107: 244254.
  • 6
    Fickert P, Zollner G, Fuchsbichler A, Stumptner C, Weiglein AH, Lammert F, Marschall HU, et al. Ursodeoxycholic acid aggravates bile infarcts in bile duct-ligated and Mdr2 knockout mice via disruption of cholangioles. Gastroenterology 2002; 123: 12381251.
  • 7
    Rosmorduc O, Hermelin B, Poupon R. MDR3 gene defect in adults with symptomatic intrahepatic and gallbladder cholesterol cholelithiasis. Gastroenterology 2001; 120: 14591467.
  • 8
    Rosmorduc O, Hermelin B, Boelle PY, Parc R, Taboury J, Poupon R. ABCB4 gene mutation-associated cholelithiasis in adults. Gastroenterology 2003; 125: 452459.
  • 9
    Konikoff FM, Chung DS, Donovan JM, Small DM, Carey MC. Filamentous, helical, and tubular microstructures during cholesterol crystallization from bile. Evidence that cholesterol does not nucleate classic monohydrate plates. J Clin Invest 1992; 90: 11551160.
  • 10
    Wang DQ-H, Carey MC. Complete mapping of crystallization pathways during cholesterol precipitation from model bile: influence of physical-chemical variables of pathophysiological relevance and identification of a stable liquid crystalline state in cold, dilute and hydrophilic bile salt-containing systems. J Lipid Res 1996; 37: 606630.
  • 11
    Wang DQ-H, Carey MC. Characterization of crystallization pathways during cholesterol precipitation from human gallbladder biles: identical pathways to corresponding model biles with three predominating sequences. J Lipid Res 1996; 37: 25392549.
  • 12
    Wang DQ-H, Paigen B, Carey MC. Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: physical-chemistry of gallbladder bile. J Lipid Res 1997; 38: 13951411.
  • 13
    Van Nieuwkerk CMJ, Oude Elferink RPJ, Groen AK, Ottenhoff R, Tytgat GNJ, Dingemans KP, van den Bergh Weerman MA, et al. Effects of ursodeoxycholate and cholate feeding on liver disease in FVB mice with a disrupted mdr2 P-glycoprotein gene. Gastroenterology 1996; 111: 165171.
  • 14
    Van Nieuwkerk CMJ, Groen AK, Ottenhoff R, van Wijland M, van den Bergh Weerman MA, Tytgat GNJ, Offerhaus JJA, Oude Elferink RPJ. The role of bile salt composition in liver pathology of mdr2 (−/−) mice: differences between males and females. J Hepatol 1997; 26: 138145.
  • 15
    Crawford AR, Smith AJ, Hatch VC, Oude Elferink RPJ, Borst P, Crawford JM. Hepatic secretion of phospholipid vesicles in the mouse critically depends on mdr2 or MDR3 P-glycoprotein expression. J Clin Invest 1997; 100: 25622567.
  • 16
    Oude Elferink RPJ, Ottenhoff R, van Wijland M, Frijters CMG, van Nieuwkerk C, Groen AK. Uncoupling of biliary phospholipid and cholesterol secretion in mice with reduced expression of mdr2 P-glycoprotein. J Lipid Res 1996; 37: 10651075.
  • 17
    Lammert F, Wang DQ-H, Paigen B, Carey MC. Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: integrated activities of hepatic lipid regulatory enzymes. J Lipid Res 1999; 40: 20802090.
  • 18
    Wang DQ-H, Lammert F, Paigen B, Carey MC. Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: pathophysiology of biliary lipid secretion. J Lipid Res 1999; 40: 20662079.
  • 19
    Hillebrandt S, Goos C, Matern S, Lammert F. Genome-wide analysis of hepatic fibrosis in inbred mice identifies the susceptibility locus Hfib1 on chromosome 15. Gastroenterology 2002; 123: 20412051.
  • 20
    Hesse A, Molt K. Technik der infrarotspektroskopischen Harnsteinanalyse. J Clin Chem Clin Biochem 1982; 20: 861873.
  • 21
    Trotman BW, Morris TA, Sanchez HM, Soloway RD, Ostrow JD. Pigment versus cholesterol cholelithiasis: identification and quantification by infrared spectroscopy. Gastroenterology 1977; 72: 495498.
  • 22
    Edwards JD, Adams WD, Halpert B. Infrared spectrums of human gallstones. Am J Clin Path 1958: 236238.
  • 23
    Konikoff FM, Carey MC. Cholesterol crystallization from a dilute bile salt-rich model bile. J Crystal Growth 1994; 144: 7986.
  • 24
    Konikoff FM, Laufer H, Messer G, Gilat T. Monitoring cholesterol crystallization from lithogenic model bile by time-lapse density gradient ultracentrifugation. J Hepatol 1997; 26: 703710.
  • 25
    Loomis CR, Shipley GG, Small DM. The phase behaviour of hydrated cholesterol. J Lipid Res 1979; 20: 525535.
  • 26
    Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959; 37: 911917.
  • 27
    Bartlett GR. Phosphorus assay in column chromatography. J Biol Chem 1959; 234: 466468.
  • 28
    Gurantz D, Laker MF, Hofmann AF. Enzymatic measurement of choline-containing phospholipids in bile. J Lipid Res 1981; 22: 373376.
  • 29
    Carey MC. Critical tables for calculating the cholesterol saturation of native bile. J Lipid Res 1978; 19: 945955.
  • 30
    Lehmann WD, Köster M, Erben G, Keppler D. Characterization and quantification of rat bile phosphatidylcholine by electrospray-tandem mass spectrometry. Anal Biochem 1997; 246: 102110.
  • 31
    Brügger B, Erben G, Sandhoff R, Wieland FT, Lehmann WD. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry. Proc Natl Acad Sci U S A 1997; 94: 23392344.
  • 32
    Konikoff FM, Cohen DE, Carey MC. Filamentous crystallization of cholesterol and its dependence on lecithin species in bile. Mol Cryst Liq Cryst 1994; 248: 291296.
  • 33
    Rapaport H, Kuzmenko I, Lafont S, Kjaer K, Howes PB, Als-Nielsen J, Lahav M, et al. Cholesterol monohydrate nucleation in ultrathin films on water. Biophys J 2001; 81: 27292736.
  • 34
    Bonnett R, Davies JE, Hursthouse MB. Structure of bilirubin. Nature 1976; 262: 327328.
  • 35
    Carey MC, Small DM. The characteristics of mixed micellar solutions with particular reference to bile. Am J Med 1970; 49: 590608.
  • 36
    Carey MC, Montet JC, Phillips MC, Armstrong MA, Mazer NA. Thermodynamic and molecular basis for dissimilar cholesterol-solubilizing capacities by micellar solutions of bile salts: cases of sodium chenodeoxycholate and sodium ursodeoxycholate and their glycine and taurine conjugates. Biochemistry 1981; 20: 36373648.
  • 37
    Wang DQ-H, Tazuma S. Effect of β-muricholic acid on the prevention and dissolution of cholesterol gallstones in C57L/J mice. J Lipid Res 2002; 43: 19601968.
  • 38
    Heuman DM. Quantitative estimation of the hydrophilic-hydrophobic balance of mixed bile salt solutions. J Lipid Res 1989; 30: 719730.
  • 39
    Hay DW, Cahalane MJ, Timofeyeva N, Carey MC. Molecular species of lecithins in human gallbladder biles. J Lipid Res 1993; 34: 759768.
  • 40
    Moschetta A, van Berge-Henegouwen GP, Portincasa P, Palasciano G, van Erpecum KJ. Cholesterol crystallization in model biles: effects of bile salt and phospholipid species composition. J Lipid Res 2001; 42: 12731281.
  • 41
    Lammert F, Marschall HU, Glantz A, Matern S. Intrahepatic cholestasis of pregnancy: molecular pathogenesis, diagnosis and management. J Hepatol 2000; 33: 10121021.
  • 42
    Shoda J, Oda K, Suzuki H, Sugiyama Y, Ito K, Cohen DE, Feng L, et al. Etiologic significance of defects in cholesterol, phospholipid, and bile acid metabolism in the liver of patients with intrahepatic calculi. HEPATOLOGY 2001; 33: 11941205.
  • 43
    Fracchia M, Pellegrino S, Secreto P, Gallo L, Masoero G, Pera A, Galatola G. Biliary lipid composition in cholesterol microlithiasis. Gut 2001; 48: 702706.
  • 44
    Shaffer EA, Small DM. Biliary lipid secretion in cholesterol gallstone disease. The effect of cholecystectomy and obesity. J Clin Invest 1977; 59: 828840.
  • 45
    Landi K, Sinard J, Crawford JM, Topazian M. Cholesterol crystal morphology in acalculous gallbladder disease. J Clin Gastroenterol 2003; 36: 364366.
  • 46
    Carey MC, LaMont JT. Cholesterol gallstone formation. 1. Physical-chemistry of bile and biliary lipid secretion. Prog Liver Dis 1992; 10: 139163.
  • 47
    Small DM. Role of ABC transporters in secretion of cholesterol from liver into bile. Proc Natl Acad Sci U S A 2003; 100: 46.
  • 48
    Schwab RG, Theis JH. Annual cyclicity of gallstone prevalence in deer mice (Peromyscus maniculatus gambelii). J Wildlife Dis 1989; 25: 462468.
  • 49
    Holzbach RT, Marsh M, Hallberg MC. The effect of pregnancy on lipid composition of guinea pig gallbladder bile. Gastroenterology 1971; 60: 288293.
  • 50
    Jenkins SA. Biliary lipids, bile acids and gallstone formation in hypovitaminotic C guinea pigs. Br J Nutr 1978; 40: 317322.
  • 51
    Snowball S, de Ranter C, Fevery J. Lincomycin treatment of guinea pigs causes formation of pigmented phosphate containing gallbladder sludge and stones. J Hepatol 1989; 9: 159166.
  • 52
    Taylor DR, Crowther RS, Cozart JC, Sharrock P, Wu J, Soloway RD. Calcium carbonate in cholesterol gallstones: polymorphism, distribution, and hypotheses about pathogenesis. HEPATOLOGY 1995; 22: 488496.
  • 53
    Konikoff FM, Cohen DE, Carey MC. Phospholipid molecular species influence crystal habits and transition sequences of metastable intermediates during cholesterol crystallization from bile salt-rich model bile. J Lipid Res 1994; 35: 6070.
  • 54
    Sakomoto M, Tazuma S, Chayama K. Less hydrophobic phosphatidylcholine species simplify biliary vesicle morphology, but induce bile metastability with a broad spectrum of crystal forms. Biochem J 2002; 362: 105112.
  • 55
    Van Erpecum KJ, Portincasa P, Gadellaa M, van de Heijning BJM, Renooij W, van Berge-Henegouwen GP. Effects of bile salt hydrophobicity on nucleation behaviour of cholesterol crystals in model bile. Eur J Clin Invest 1996; 26: 602608.