Bile salt hydrophobicity controls vesicle secretion and transformations in native bile DAVID

E. COHEN,

LESLIE

S. LEIGHTON,

AND MARTIN

rates

C. CAREY

Departments of Medicine and Physiology and Biophysics, Harvard Medical School, Brigham and Women’s Hospital, and Harvard Digestive Diseases Center, Boston, Massachusetts 02115 Cohen, David E., Leslie S. Leighton, and Martin C. Carey. Bile salt hydrophobicity controls vesicle secretionrates and transformations in native bile. Am. J. Physiol. 263 (Gastrointest. Liver PhysioZ. 26): G386-G395, 1992.-After drainage

of the bile salt pool, we infused unanesthetized bile fistula prairie dogs (Cynomys Zudovicianus) intravenously with taurineconjugated chenodeoxycholate (TCDC), cholate (TC), ursodeoxycholate (TUDC), and ursocholate (TUC) in concentrations that attained >94% enrichment of biliary bile salts. With decreasesin bile salt hydrophobicity, maximum steady state lecithin and to a lesserextent cholesterol secretionrates decreased in the rank order, TCDC > TC > TUDC > TUC. By phase analysis, TCDC-rich and TC-rich biles plotted inside their respective micellar zones, whereas TUDC-rich and TUC-rich biles plotted outside and were so-called“supersaturated” with cholesterol. Quasi-elastic light scattering and electron microscopy, when performed within 30 min of collection, revealed unilamellar vesiclesin all biles. By 24 h, vesiclesin TCDC-rich and TC-rich biles had dissolved into mixed micelles, whereas vesiclesin TUDC-rich biles formed mixed micellesplus multilamellar liquid crystals, and vesiclesin TUC-rich biles formed multilamellar liquid crystals exclusively. Because cholesterol/ phospholipid molar ratios of multilamellar liquid crystals were 5 1, cholesterol monohydrate crystals did not form in these biles. We conclude that, despite drastic alterations in bile salt detergency, unilamellar vesiclesare the final common pathway for lecithin and cholesterol secretion into bile. During equilibration of bile, the fate of unilamellar vesiclesmay be micellar, micellar plus liquid crystalline, or liquid crystalline only depending on the detergency (i.e., hydrophobic-hydrophilic balance)of the secretedbile salt. lecithin; cholesterol; quasi-elastic light scattering; electron microscopy; polarizing light microscopy; gel filtration; phase analysis SALTS PROMOTE the biliary secretion of lecithin and cholesterol and participate in solubilizing these lipids in bile (5). It is believed that, at the canalicular level, bile salt monomers and lecithin-cholesterol vesicles are secreted independently into bile (12). As bile is concentrated within the biliary tree, bile salts and vesicles interact to form mixed micelles in cholesterol unsaturated biles (10, 11) or saturated mixed micelles plus cholesterol-enriched vesicles in supersaturated biles (11). Although it has been suggested that biliary lipid secretion rates vary dramatically as functions of the hydrophobichydrophilic balance of the secreted bile salt species (3, 20), neither the mechanism(s) whereby common bile salts of different hydrophobicities promote biliary lecithin and cholesterol secretion rates nor the influence of bile salt hydrophobicity on the physical chemistry of native biles has been explored systematically. We designed the present study to examine the influence of bile salt species covering a wide range of hydrophobicities (1, 22) on biliary lipid secretion and timedependent physical states of bile in the bile fistula

BILE

G386

0193-1857/92

$2.00

Copyright

prairie dog. After depletion of the endogenous bile salt pool, intravenous infusions of 3cu,7cu-dihydroxy-5P-cholanoate (taurochenodeoxycholate; TCDC), 3cw,7ar,12a-trihydroxy@?-cholanoate (taurocholate; TC), 3a,7@-dihydroxy-5P-cholanoate (tauroursodeoxycholate; TUDC), and 3a,7P,12a-trihydroxy-5P-cholanoate (tauroursocholate; TUC) produced hepatic bile that was essentially replaced with the infused bile salt species. Lecithin and cholesterol were secreted as unilamellar vesicles irrespective of bile salt hydrophobicity, and the rates varied positively with bile salt hydrophobicity. Because increasing bile salt hydrophobicity promoted biliary secretion of lecithin in excess of cholesterol molecules, vesicle cholesterol/lecithin ratios were inversely proportional to bile salt hydrophobicity. In biles containing the more hydrophobic TCDC and TC species, bile salts dissolved secreted vesicles into mixed micelles during equilibration, whereas in biles with hydrophilic bile salts, vesicles aggregated in part (TUDC-rich bile) or completely (TUC-rich bile) to form multilamellar liquid crystalline phases. EXPERIMENTAL Materials

PROCEDURES

Sodium saltsof TCDC, TC, TUDC (Calbiochem,San Diego, CA), and TUC (a generousgift of Dr. G. Salen,Veterans Affairs Medical Center, East Orange, NJ) were purified, and each gave single spots on thin-layer chromatography after 200-pg sample application (-98% pure). Grade I egglecithin (Lipid Products, South Nutfield, Surrey, UK) was >99% pure by high-performance liquid chromatography (HPLC; seeRef. 11) and thinlayer chromatography (200 ,ug application). Cholesterol was obtained from Nu-Check Prep (Elysian, MN) and found to be 99% pure by gaschromatography. Tris(hydroxymethyl)aminomethane (Tris) l HCl was obtained from Sigma (St. Louis, MO), and all other chemicals and solvents were American Chemical Society (ACS) or reagent grade purity (Fisher Scientific, Medford, MA). ACS quality NaCl wasroastedat 600°C for 4 h to oxidize and remove organic impurities. Pyrex-brand glassware was alkali washed for 24 h (EtOH-2 M KOH, 1:1, vol:vol) followed by 24 h acid washing (1 M HNO,) and rinsed thoroughly with purified water. Water was filtered, ion exchanged, and glass distilled (Corning Glass Works, Corning, NY). Animals

Male black-tailed prairie dogs(Cynomys ludovicianus) weighing 1.0 t 0.2 kg (Prairie Dog Supply, Minneapolis, MN) were maintained on Purina guinea pig chow and provided water ad libitum. After an overnight fast, anesthesiawas administered according to Roslyn et al. (29). Briefly, 20 mg/kg of Xylazine (Haver-Lockhart, Shawnee, KS) was injected intramuscularly followed 20 min later by a 100 mg/kg im injection of ketamine (Parke-Davis, Morris Plains, NJ). For surgery lasting longer than 1 h, additional 50-mg/kg dosesof ketamine were administered at 30-min intervals. A femoral vein wascannulated with

0 1992 the American

Physiological

Society

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PE-50 polyethylene tubing (Clay Adams, Becton-Dickinson, Parsippany, NJ). Upon securing the cannula with silk sutures, an intravenous infusion of 150 mM NaCl at 1 ml/h (Harvard Infusion Pump, Harvard Apparatus, South Natick, MA) was begun and maintained for the duration of each experiment. Through an abdominal incision, the cystic duct was ligated, and gallbladder bile was aspirated. A PEG0 polyethylene cannula was inserted in the common bile duct and secured with silk sutures, thereby completely diverting bile flow for collection. The bile duct cannula was externalized, the abdominal incision closed, and the prairie dog placed in a steel restraining cage with access to guinea pig chow and water. During surgery and throughout each experiment, body temperature was maintained by an infrared lamp at 37 t 1 “C as monitored by a rectal probe (YSI Telethermometer, Yellow Springs Instrument, Yellow Springs, OH). Methods Model biles. Model biles were used to establish equilibrium micellar zones for phase analysis, and intermicellar bile salt concentrations were measured for Superose 6 gel filtration of native biles, respectively (see below). After coprecipitation from stock solutions (CHC1,/MeOH 1:1, vol:vol), dried bile salt/ lecithin/cholesterol films were resuspended in aqueous solution (0.15 M NaCl, -pH 7) that included 3.0 mM NaN3 as an antimicrobial agent (8). Before determination of the equilibrium physical-chemical state, model biles were incubated at 37°C under an atmosphere of argon. Quasi-elastic light scattering (&LS). QLS measurements of model and native biles were performed employing an apparatus detailed elsewhere (14). Briefly, the apparatus consisted of a Spectra Physics (Mountain View, CA) model 164 argon ion laser tuned to 5,145 A, a 64-channel Langley-Ford (Amherst, MA) model 1096 correlator, and a home-built temperature-controlled sample holder based on a Peltier-effect module (model CP2-31-06L, Melcore, Trenton, NJ). For data analysis, a Digital (Maynard, MA) Pro 350 model computer was interfaced with the correlator. Measurements were performed at a scattering angle of 90” with samples maintained at 37°C. We employed two methods to analyze intensity autocorrelation functions of scattered laser light. 1) Cumulants analysis gave the mean diffusion coefficient of all particles in bile and the variance of particle sizes (14). 2) When variance (a polydispersity index) exceeded 50%) multiexponential analysis (14) was employed. In general, the latter demonstrated that two components (i.e., the presence of two discrete particle populations) provided the best computer fits to the light-scattering data, and analysis yielded mean diffusion coefficients as well as relative concentrations (wt/wt) of both micelle and vesicle populations with accuracies of &lo% (D. E. Cohen and M.C. Carey, unpublished observations). Mean hydrodynamic radii (&) of particle populations were calculated from the weighted or individual diffusion coefficients using the Stokes-Einstein relationship (14). When present in biles, multilamellar liquid crystals, which were generally too large (Eh > 10,000 A) to be resolved by QLS (14), were removed by ultracentrifugal separation before QLS analysis (see below). Superose 6 gel filtration. To fractionate biles, a prepacked and factory calibrated (i.e., with specified void and total volumes) Pharmacia (Piscataway, NJ) HR10/30 Superose 6 column was used (13). Samples (0.5 ml) were applied to the column with a Pharmacia P-500 pump in continuous mode delivering eluant at a flow rate of 30 ml/h. Fractions (1.0 ml) were collected continuously using a Pharmacia-LKB Frac-100 fraction collector. When native prairie dog biles were fractionated, columns were preequilibrated and eluted with buffer containing 0.15 M NaCl, 0.020 M Tris HCl (pH 8.5), 3.0 mM NaN,, and 5.0 mM

AND

BILIARY

LIPIDS

G387

sodium ascorbate as antioxidant. Depending on the infused bile salt species, we added to the buffer the appropriate intermicellar concentrations of TCDC (2.0 mM), TC (7.5 mM), and TUDC (6.0 mM) to preserve vesicle and mixed micellar integrity (17). Because in TUC-rich biles a total concentration of 8.0 mM TUC was secreted but was insufficient to solubilize lecithin or cholesterol in model biles (see RESULTS), the column was preequilibrated and eluted with this TUC concentration. In preliminary experiments, the intermicellar concentrations of TCDC, TC, and TUDC were determined as detailed elsewhere (13). Briefly, cholesterol supersaturated model biles (3 g/dl, 0.15 M NaCl, 3.0 mM NaN,, pH -7) were prepared with relative lipid compositions of TCDC/lecithin/cholesterol 63:27:10, TC/ lecithin/cholesterol 63:27:10, and TUDC/lecithin/cholesterol 71:24:5. We prepared a series of trial eluants containing a range of bile salt concentrations (in 0.15 M NaCl, 3.0 mM NaN, at from well above to slightly PH -7) that varied systematically below estimates of their critical micellar concentrations (7, 13). Portions (0.5 ml) of each model bile were fractionated into vesicles and mixed micelles using the trial eluants. The correct intermicellar bile salt concentration was identified as the eluant bile salt concentration that yielded both high-resolution separation of vesicles and mixed micelles as well as distribution of cholesterol between particles as predicted by the appropriate ternary bile salt/lecithin/cholesterol phase diagram (see PHASE ANALYSIS; also see Ref. 13). Because studies in model systems (17) have shown that intermicellar concentrations do not vary appreciably within the range of total and relative lipid compositions observed in prairie dog biles, a single intermicellar concentration for each bile salt served as a close approximation to the true intermicellar concentrations (17) for the purposes of the present study. Negative stain eLectron microscopy. To elucidate structures of biliary lipid aggregates, prairie dog biles were fixed and stained using 1% phosphotungstic acid on Formvar-coated grids as previously described (10). Samples were then viewed with a Philips 300 electron microscope (Philips Electronic Instruments, Mahwah, NJ) and photographed. Polarizing light microscopy. Model and native biles were examined by polarizing light microscopy (Photomicroscope III, Zeiss, Thornwood, New York). A few microliters of well-mixed samples were placed between a glass slide and cover slip. Multilamellar liquid crystals containing cholesterol and lecithin were identified as birefringent Maltese crosses (focal tonics), which deformed on compression, whereas cholesterol monohydrate crystals demonstrated platelike structures with a characteristic notched corner (8). Ultracentrifugation. To isolate multilamellar liquid crystalline phases from vesicles and mixed micelles, prairie dog biles were ultracentrifuged (lOO,OOO-160,000 g at 37°C) for 16-36 h (Beckman model L5-65 ultracentrifuge, SW 50.1 rotor, Fullerton, CA). In model systems, these centrifugal forces neither induce sedimentation of mixed micelles (32) nor aggregation of unilamellar vesicles (24). When present, multilamellar liquid crystalline phases floated as thin layers of creamy appearance that were carefully sampled using a glass transfer pipette. Experimental

Design

Biliary lipid secretion rates. After overnight drainage of the endogenous bile salt pool (which contained -92 mol/lOO mol TC and -5 mol/lOO mol TCDC as determined by HPLC; see below), individual prairie dogs were infused via their femoral veins with TCDC (n = 3), TC (n = 4), TUDC (n = 2), or TUC (n = 2). Infusion rates were increased stepwise from IO to 1,000 ,umol h-l. kg body wt-l over a 6- to 8-h period in 30- to 90-min intervals. Bile was collected continuously in 15- to 30-min fractions into preweighed test tubes with the use of a fraction collector (model 7000 Ultrorac, LKB, Stockholm, Sweden), and l

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G388

BILE

SALT

HYDROPHOBICITY

final weights were obtained. Because hepatic bile density is - 1, bile volumes were assumed to be equivalent to weights. Biliary lipid compositions in all fractions and bile salt species in representative fractions were determined (see below). Bile salt, lecithin, and cholesterol secretions rates (in pmol kg-l h-l) were calculated as the products of lipid concentration and bile flow rates. Physical states of biliary lipids. PHASE ANALYSIS. We determined the micellar phase limits of physiologically relevant model biles containing egg yolk lecithin, cholesterol, and either TCDC, TC, TUDC, or TUC. We chose a total lipid concentration of 3 g/d1 because it approximated the average total lipid concentration of prairie dog hepatic biles (range 0.5-7.0 g/dl) after individual bile salt infusions. In the case of TC, we used published experimental values (8) for the micellar zone of TC (3 g/dl, 0.15 M NaCl, 37°C pH -7), and for TCDC, TUDC, and TUC, micellar zones were established according to phase equilibria procedures (8, 24, 32). Briefly, model biles (3 g/dl, 0.15 M NaCl, pH -7) were prepared with relative lipid compositions that spanned the physiologically relevant region of the ternary bile salt-lecithin-cholesterol phase diagram (i.e., mol/lOO mol bile salt > 60) and were incubated at 37°C under an atmosphere of argon in the dark with periodic vortex mixing. At daily intervals, lipid mixtures in sealed tubes were illuminated with the use of a high-intensity light source, and their appearances were recorded. After an equilibration period (IO-14 days), well-mixed samples were examined by polarizing light microscopy and by QLS. Model bile compositions were considered to fall within the micellar zone of the phase diagram if solutions were optically clear, devoid of liquid crystals or cholesterol monohydrate crystals by polarizing light microscopy, and, by QLS, contained a single population of pa:title sizes consistent with mixed micelles (Eh - 20-60A; see Ref. 24). Repeat analyses over a several month period revealed no change in the micellar phase limits, suggesting that micellar solutions had reached equilibrium. Phase analysis of native biles was performed by plotting on triangular coordinates relative compositions (i.e., mol/lOO mol bile salts vs. mol/lOO mol lecithin vs. mol/lOO mol cholesterol) of prairie dog biles with the micellar phase boundary of the ternary systems containing the same bile salt species. Relative lipid compositions of bile samples employed to define biliary lipid secretion rates were calculated as: mol/lOO mol biliary lipid = 100 X its respective secretion rate/(bile salt secretion rate + lecithin secretion rate + cholesterol secretion rate). Although phase analysis of the three lipids reliably predicted biliary cholesterol supersaturation, such analyses could not, by definition, predict metastable physical-chemical states in cholesterol supersaturated biles (4). PHYSICAL-CHEMICAL ANALYSIS OF NATIVE BILES. In a separate set of experiments, after overnight depletion of the endogenous bile salt pool, individual prairie dogs were infused with TCDC (n = 2), TC (n = 2), TUDC (n = 2), or TUC (n = 2) at 200 prnol. kg-l. h-l for 6-h periods. During the 6th hour of infusion, negative stain electron microscopy and polarizing light microscopy were performed on biles within 30 min of collection, and this was repeated after 24 h equilibration under argon at 37°C. In preliminary experiments in this work and in previous studies of rat biles (IO), we discovered a population of proteinglycoprotein aggregates in bile. These aggregates persisted after dissolution of vesicles and produced QLS sizes approximately equal to those of unilamellar vesicles (& - 300-7OOA). Because the sizes of these proteins varied unpredictably from animal to animal but were constant over time for a single animal, we assessed the influence of bile salt species on vesicle dissolution against this protein aggregate background in a single prairie dog. After overnight depletion of the endoeenous bile salt nool. l

l

AND

BILIARY

LIPIDS

TCDC, TC, TUDC, and TUC were serially infused at a rate of 10 prnol. kg-l h-l into the same prairie dog for 6-h periods. Between infusions of different bile salts, HPLC analysis revealed that a 6-h washout period was sufficient to eliminate the previously infused bile salt species. QLS measurements of biles were performed within 30 min of collection during the 6th hour of infusion and again after a 24-h equilibration period. A liquid crystalline phase present in TUDC-rich and TUC-rich biles was isolated by ultracentrifugal separation and analyzed for lipid composition (see Analytical Procedures). Lipid phases coexisting in the infranatant after ultracentrifugation were separated by Superose 6 gel filtration. l

Analytical

Procedures

Lecithin concentrations were determined from inorganic phosphorus (8)) cholesterol concentrations were measured by the cholesterol oxidase method (19) or by gas chromatography (Shimadzu model GCSa, Kyoto, Japan) (IO), and bile salt concentrations were measured by the 3a-hydroxysteroid dehydrogenase method (8). Biliary bile salt species were analyzed by HPLC (Altex model 1 IOA, Beckman Instruments, Fullerton, CA; see Ref. I). Protein analysis employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with periodic acid-Schiff reagent and Coomassie blue as described previously (10). Mathematical

Modeling

of

Biliary

Lipid

Secretion

Biliary lecithin and cholesterol secretion rates increased curvilinearly with increasing bile salt secretion rates and approached limiting values (see RESULTS). On this basis, biliary lecithin secretion rates (L,,,) and cholesterol secretion rates (Ch,,,) were represented as hyperbolic functions of bile salt secretion rates (BS,,,; developed in Ref. 25). The general form of the equations are L set

L max

BSsec

= al + BS,,, Ch max

Chsec

BSsec

= a2

+

BSsec

(14

um

where L,,, and Ch,,, are maximum secretion rates of lecithin and cholesterol, respectively, and values of a1 and cy2are secretion rates of bile salts at one-half maximum secretion rates of lecithin (Eq. IA) or cholesterol (Eq. 1 B), respectively. To facilitate curve-fitting and statistical comparisons, the hyperbolic functions in Eqs. IA and IB were reexpressed using linear transformations analogous to those in enzyme kinetics (23) BSsec -= L set BSsec -=Chsec

BSsec

p+-

L max

max

BSsec

a2

Chmax

+

(24 (2B)

Ch,,,

In this manner, L,,, and Ch,,, were calculated from slopes determined by linear least squares fitting of experimental data, and values of al and a2 were calculated from the intercept values. At low bile salt secretion rates (i.e., BS,,, 94%) in hepatic biles.

Biliary

Lipid

Secretion

Rates

Figure 2 displays lecithin and cholesterol secretion rates plotted as functions of the respective bile salt secretion rates. In each case, both lecithin (top) and cholesterol secretion rates (bottom) increased at low bile salt secretion rates and then leveled off. No leveling off was apparent with TCDC (Fig. ZA), because secretion rates of this bile salt X00 pmol kg-l h-l were associated with a diminution in bile flow due to bile salt hepatotoxicity. Linear regression analysis of lecithin and cholesterol secretion rates as functions of bile salt secretion rates yielded acceptable results (R > 0.60 and P < 0.001) for all bile salts except TUDC (Fig. ZC) where R = 0.46, P = 0.005 for lecithin secretion rate vs. TUDC secretion rate (top) and R = 0.18, P = 0.31 for cholesterol secretion rate vs. TUDC secretion rate (bottom). Figure 3, A and B, displays the entire TUDC data set replotted according to Eqs. ZA and 2B. In each case, highly significant linear correlations were demonstrated between TUDC secretion rates normalized for lecithin secretion rates vs. TUDC secretion rates (R = 0.97, P = 0.0001) and between TUDC secretion rates normalized for cholesterol secretion rates vs. TUDC secretion rates (R = 0.87, P = 0.0001). When all other data sets were

Statistical Analysis Biliary lipid secretion rates for prairie dogs infused with the same bile salt species were pooled before statistical analysis (20). Slopes, intercepts, and their standard deviations were obtained by linear regression analyses estimated according to the method of Johansen and Lumry (23). Analysis of variance was employed to detect differences among mean values, whereas analysis of covariance was performed for slopes of linear regressions (34). Differences between pairs of means and slopes were assessed according to the Fisher’s protected least squares differences method at a significance level of 0.05 (18). Intercept values of linear regressions were compared using a two-tailed Student’s t test (23).

l

RESULTS

Biliary Secretion of Infused Bile Salt Species

After bile duct cannulation, endogenous bile salt secretion rates fell from -40 to TC > TUDC > TUC). Because of the reported high critical micellar concentration of TUC [ ~40 mM in 0.15 M NaCl (28)], no micellar zone was obtained for 3 g/d1 TUC-egg yolk lecithin-cholesterol under the conditions of these experiments (see METHODS). Relative lipid compositions for TC-rich and TCDC-rich biles (Fig. 5, A and B) fell predominantly inside the micellar zones indicating that these biles were unsaturated with cholesterol, whereas relative lipid compositions of TUDC-rich biles plotted outside the micellar zone and hence were supersaturated with cholesterol (Fig. 5C). Because no mixed micellar zone was found for TUC-containing model bile systems (Fig. 5D), all TUC-rich biles were supersaturated with cholesterol. Figure 6 displays representative transmission electron micrographs of TCDC-, TC-, TUDC-, and TUC-rich biles negatively stained within 30 min after bile collection (see METHODS). Each micrograph demonstrates the pre!ence of particles ranging in diameter from 300 to 800 A, consistent with unilamellar vesicles (10). In addition to vesicle-sized particles, photomicrographs of TCDC-rich biles contain areas of negatively stained artifacts. After

Table 1. Biliary lipid secretion rates in bile fist&a prairie dogs after intravenous infusion of common taurine-conjugated bile salts Bile Salt Species

Bile Salt Hydrophobic Index

pmol

L maxf - kg-l - h-l

Qlt

pmol

- kg-l

- h-l

Ch max7 pmol - kg-l - h-l

a29

pmol - kg-l

CWL,

- h-l

Molar

ratio

TCDC 0.46" 16.9t1.gb 30.6k8.7" 7.0tl.3d 108t26" 0.22t0.08f TC 0.00 18.9tl.O 59.4t9.8 6.9t2.2 331t90 0.18t0.08 TUDC -0.47 8.3t0.8 28.1t9.8 2.9kl.l 20.9t25 0.41t0.16 TUC -0.94 5.2kO.6 117t27 3.8t0.5 148t35 0.67kO.15 Data (means t SD) were derived by mathematical modeling of lecithin and cholesterol secretion rates as hyperbolic functions of bile salt secretion rates. (See text for details.) L,a,, maximum lecithin secretion rate; cyl, bile salt secretion rate at which lecithin secretion rate is one-half L maxi Chrnax7 maximum cholesterol secretion rate; az, bile salt secretion rate at which cholesterol secretion rate is one-half Chma,; Ch/L, cholesterol/lecithin molar ratio; TCDC, taurochenodeoxycholate; TC, taurocholate; TUDC, tauroursodeoxycholate; TUC, tauroursocholate. a Value based on reverse phase high-performance liquid chromatography retention times of bile salts are from Ref. 22. b All values in column statistically different (P = 0.0001). c All values in column statistically different (P c 0.05) except TCDC vs. TUDC. d All values in column statistically different (P = 0.01 TUC vs. TUDC, P = 0.0001 all others). e All values in column statistically different (P < 0.05) except TCDC vs. TUC and TC vs. TUC. f All values in column statistically different (P = 0.0001) except TCDC vs. TC. Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 19, 2019.

BILE SALT HYDROPHOBICITY

AND BILIARY

G391

LIPIDS

% LECITHIN

0 1

I B

6

-

6

-

1

I

1 R. 0.63

c

R.0.92

)

0.6

% BILE SALT

f

‘il

6

0 -1

I

I

-0.5

0

BILE SALT

HYDROPHOBIC

0.5

INDEX

Fig. 4. Correlations (R) of bile salt hydrophobic index (22) with maximum lecithin secretion rates (A), maximum cholesterol secretion rates (B), cholesterol/lecithin molar ratios (C), and ratios of maximum lecithin secretion rate/o1 (D); values are means + SD. Data are replotted for all bile fistula prairie dogs from Table 1. SDS for ratios of maximum lecithin secretion rate/a1 were estimated from individual SDS according to Ref. 2. In each case, solid line represents a linear least squares fit to data. Maximum lecithin and cholesterol secretion rates as well as ratios of maximum lecithin secretion rate/o1 at low bile salt secretion rates correlate positively with increasing bile salt hydrophobicity. In contrast, biliary cholesterol/lecithin molar ratios decrease with increasing bile salt hydrophobicity. I

24 h equilibration, repeat negative stain electron microscopy of TCDC-rich and TC-rich biles failed to demonstrate vesicles, indicating their dissolution. In contrast, polarizing light microscopy revealed that vesicles coalesced to form multilamellar liquid crystals, in part in TUDC-rich biles, and completely in TUC-rich biles (data not shown). In no equilibrated TUDC-rich or TUC-rich biles were cholesterol monohydrate crystals detected . To probe further the immediate and equilibrium physical states of native biles, sizes and chemical compositions of lipid aggregates were determined for biles collected from one prairie dog after individual bile salt infusions. Immediately after bile collection, two-compo-

Fig. 5. Relative biliary lipid compositions (0) of prairie dog biles plotted on truncated triangular phase diagrams after intravenous infusions of TCDC (A), TC (B), TUDC (C), and TUC (D). In each panel, the shaded micellar zone is shown for the same bile salt species determined in ternary (bile salt, lecithin, cholesterol) model systems (3 g/dl total lipid concn, 0.15 M NaCl, 3.0 mM NaNa, 37”C, and pH -7) with symbol (0) representing micellar phase limits. Compositions of TCDC- and TCrich prairie dog biles plot inside corresponding micellar zones indicating unsaturation with cholesterol, whereas the compositions of TUDC-rich biles plot outside micellar zone and are supersaturated with cholesterol. Physical state of all biles was micellar plus vesicular (unilamellar) immediately after collection (~30 min). At equilibrium (24 h), TCDC- and TC- rich biles were micellar. TUDC-rich bile was micellar and liquid crystalline, and TUC-rich bile was liquid crystalline only: because of the high critical micellar concentration of TUC [ -40 mM (20)], no micellar phase was present.

nent QLS analyses confirmed the electron microscopic finding of vesicle: in all fresh biles. Coexisting vesicle and mixed micellar Rh values as well as concentrations (wt/ wt) were determined in TCDC-rich bile (vesicle & = 220 A, vesicles concn = 2.0%; mixed micellar &, = 30 A, mixed micellar concn = 98%), TC-rich bile (vesicle & = 375 A, vesicle concn = 2.8%; mixed micellar &, = 46 A, mixed micellar concn = 97%), and TUDC-rich bile (vesicle q-, = 510 A, vesicle concn = 16%; mixed micellar Rh = 45 A, mixed micellar concn = 84%). Because QLS revealed only a vesicle-sized population (il, = 670 A) in TUC-rich bile, an undetected but coexisting population of mixed micelles could not be excluded (14) but was highly unlikely based on phase analysis. After 24 h equilibration, repeat two-component QLS analysis of TCDC-rich and TC-rich biles revealed only traceOquantities (~0.3%) of_vesicle-siz?d particles (& = 375 A in TCDC-rich bile, Rh = 300 A in TC-rich bile) coexisting with >99.7% mixed micelles (&, = 28 A in TCDC-rich bile, & = 22 A in TC-rich bile). Superose 6 gel filtration chromatography of each bile confirmed the presence of two particle populations: Figure 7, A and C, shows that particles of mixed micellar sizes (& = 20-30 A) eluted within the included volume, whereas vesicle-sized particles (& = 250-350 A) eluted at

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G392

BILE SALT HYDROPHOBICITY

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LIPIDS

analysis, the liquid crystalline phase of TUDC-rich bile was composed of lecithin and cholesterol (molar ratio -1.0) with traces of TUDC. After complete aspiration of the liquid crystalline phase, Superose 6 gel filtration

400

t

200

10 t

Ii

06

Fig. 6. Negative stain electron micrographs of prairie dog biles after intravenous infusions of TCDC, TC, TUDC, and TUC. Each bile was negatively stained within 30 min after collection. Large unstained areas in TCDC-rich bile are artifacts (see text). Inscribed bar (TUC) represents 1,000 8, in all panels. Each micrograph demonstrates presence of unilamellar vesicles ranging in diameter from 300 to 600 A.

the void volume. As demonstrated in Fig. 7, B and D, lipid analysis of column fractions from TC-rich and TCDCrich biles revealed that all of the detectable biliary bile salts, lecithin, and cholesterol eluted as mixed micelles at equilibrium.1 In void volume fractions where vesicles would have eluted, SDS-PAGE revealed high molecular mass glycoproteins (M, 2 100 kDa) that stained with periodic acid-Schiff reagent as well as multiple small proteins that stained with Coomassie blue. In contrast to vesicle dissolution into mixed micelles in TCDC-rich and TC-rich biles, vesicles in TUDC-rich and TUC-rich biles coalesced to form multilamellar liquid crystals as evidenced by polarizing light microscopy after 24 h equilibration. After ultracentrifugal separation and i To test whether void volume fractions contained vesicles at concentrations too low to be detected by lipid analysis, we attempted to dissolve the large aggregates with exogenous bile salts (10). We added 100 mM TCDC or TC to void volume fractions from Fig. 7, A and C, respectively, and repeated the QLS analysis. In each case, the larger particle populations did not dissolve proving that they were not vesicles (10).

,J

0

EL U T/OiV VOLUME fmN Fig. 7. Superose 6 gel filtration analysis of equilibrated TCDC-, TC-, and TUDC-rich prairie dog biles fractionated with eluant (0.15 M NaCI, 3.0 mM NaN3) containing (in mM) 2.0 TCDC (A and B), 7.5 TC (C and D), and 6.0 TUDC (E and F). For each bile salt species, values of mean hydrodynamic radii (&; A) and variance (0) (A, C and E) and concentrations of bile salt t 100 (0), lecithin (o), and cholesterol (A) (B, D and F) are plotted as functions of elution volume. Void and total volumes (V, and V,, respectively) are indicated by arrows. Before fractionation, lipid compositions of TCDC-rich bile (2.0 g/dl, TCDC/lecithin/ cholesterol 73:24:3) and TC-rich bile (1.7 g/dl, TC/lecithin/cholesterol 71:26:3) plotted within micellar zones of Fig. 5, A and B, respectively, whereas composition of TUDC-rich bile (1.0 g/dl, TUDC/lecithin/ cholesterol 66:28:6) fell outside micellar zone of Fig. 5C. After equilibration and fractionation, all biliary lipids in TCDC- and TC-rich biles elute as mixed micelles, with only trace quantities of larger proteinglycoprotein aggregates at V, (see text for details). Although most biliary lipids in TUDC-rich bile are present as mixed micelles (and multilamellar liquid crystals that were removed by ultracentrifugation before gel filtration, see text), a small population of cholesterol-lecithin vesicles coelutes with protein-glycoprotein aggregates at V,. Because equilibrated TUC-rich bile (0.5 g/dl, TUG/lecithin/cholesterol 85:11:4) contained only multilamellar liquid crystals and neither mixed micelles nor vesicles, no lecithin or cholesterol was eluted by gel filtration after removal of multilamellar liquid crystals by ultracentrifugation (data not shown).

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chromatography of the infranatant (Fig. 7, E and F) revealed that cholesterol-lecithin ovesicles accounted for - 1% of lipids (& = 230-430 A) while the remainder eluted as mixed micelles (& = 32-38 A). Analysis of void volume fractions by SDS-PAGE revealed the same glycoprotein-protein complex as characterized above. The centrifugally isolated multilamellar liquid crystalline phase of TUC-rich bile was principally composed of cholesterol and lecithin in a ratio of -0.8, but, in the TUC-rich infranatant, only traces of lecithin or cholesterol (~1% of total) were present. QLS analysis of the infranatant revealed a single particle population with R h = 330 A, which was shown by Superose 6 gel filtration and SDS-PAGE to be composed of a glycoprotein-protein complex (Rh = 370-480 A): No vesicles, mixed micelles, or simple micelles were detected (data not shown). DISCUSSION

The mechanisms by which bile salts promote biliary lecithin and cholesterol secretion and their role in lipid aggregation during bile formation remain incompletely understood. We therefore designed this study to characterize the influence of bile salts with a broad range of distinct physical-chemical characteristics on biliary lipid secretion rates as well as the immediate and equilibrium physical states of biliary lipids in native prairie dog biles. The principal findings were the following: 1) Lecithin and cholesterol secretion rates were hyperbolic functions of bile salt secretion rates over a wide range of bile salt hydrophobicities and varied positively in proportion to bile salt hydrophobicity. 2) Regardless of bile salt hydrophobicity or micelle formation, bile salts promoted vesicular secretion of lecithin and cholesterol into bile. 3) Secreted vesicles dissolved into mixed mice11.es in biles enriched with more hydrophobic bile salts but aggregated to form mixed micelles plus multilamellar liquid crystals in TUDC-rich biles and liquid crystals only in TUC-rich biles. We discuss each of these findings in the context of relevant in vitro and in vivo studies. Influences of bile salt structure on secretion rates of lecithin and cholesterol into bile have been studied in rat (3), cat (31), dog (26), hamster (20), and human (9). In all species, variations in lecithin and cholesterol secretion rates have been attributed in part to bile salt structure. Two studies (3,20) systematically analyzed a range of bile salt species sufficient to establish lipid secretory relationships based on the p hysical- ,chemical properties of the bile salt species. In bile fistula rats infused intravenously with bile salts, Bilhartz and Dietschy (3) reported that, at low bile salt secretion rates, lecithin and cholesterol secretion rates increased linearly, and they found that coupling of lecithin and cholesterol to bile salts increased in proportion to bile salt hydrophobicity. After duodenal infusion of different bile salts in hamsters, Gurantz and Hofmann (20) observed linear relationships between bile salt secretion rates and secretion rates of lecithin and cholesterol and an inverse relationship between these rates and critical micellar concentrations of the infused bile salts. Because TUDC promoted greater biliary cholesterol and lecithin secretion than TC, biliary lipid secretion rates did not vary monotonically as functions of bile salt hydro-

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phobicity as was found in the present work (see hydrophobic indexes in Table 1). This may be accounted for by the experimental design employed by these authors in that endogenous bile salt pools of hamsters were not drained before study and therefore were incompletely replaced with the infused bile salt species. Nevertheless for taurine-conjugated bile salts, our reanalysis of the data in Ref. 20 reveals a positive correlation be tween bile salt hydroph .obic index and lecithin (R = 0.79) and ch.olesterol (R = 0.74) secretion rates. In the present study, lecithin and cholesterol secretion rates varied nonlinearly over a wide range of bile salt secretion rates (Fig. 2) and were modeled as rectangular hyperbolas according to Eqs. 1 and 2. This model allowed us to estimate, at low bile salt secretion rates, slopes of the “linear” portions of the lecithin (maximum lecithin secretion rate/al) and cholesterol (maximum cholesterol secretion rate/aZ) secretion curves. In accordance with Bilhartz and Dietschy (3), we found that lecithin coupling to bile salt secretion at low bile salt secretion rates increased with bile salt hydrophobicity (Fig. 40), but we found no correlation between cholesterol coupling and bile salt hydrophobicity (not shown) possibly because our experimental approach was relatively insensitive to small changes in cholesterol secretion rates at the lower end of the bile salt secretion curve. Our model extended results at low secretion rates to include maximum lecithin and cholesterol secretion rates. At high bile salt secretion rates, both maximum lecithin and cholesterol secretion rates increased with increases in bile salt hydrophobic index (Fig. 4, A and B). This result is consistent with observations in model systems that elution rates of lecithin and cholesterol from model membranes adsorbed to Millipore filters correlate positively with bile salt hydrophobicity (30). A stronger bile salt dependency on lecithin compared with cholesterol secretion rates (i .e., steeper slopes of linear least squares fit in Fig. 4A compared with Fig. 4B) explains decreases in molar cholesterol/lecithin ratios observed with increases in bile salt hydrophobic index (Table 1 and Fig. 4C). To gain insights into mechanisms whereby bile salts promote lecithin and cholesterol secretion into bile, we analyzed the physical states of biliary lipids immediately after bile collection. Analysis of biles enriched with each of the four bile salt species revealed unilamellar vesicles by negative stain electron microscopy (Fig. 6) and QLS. Because the relative lipid compositions of TCDC-rich and TC-rich biles fell within the micellar zone of their respective phase diagrams (Fig. 5, A and B), phase analysis predicted that, at equilibrium, these biles should contain only micelles. Indeed, the presence of vesicles in the fresh biles is consistent with the hypothesis that lecithin and cholesterol are cosecreted into bile as vesicles (10, 33), and as previously observed in cholesterol unsaturated rat biles (lo), the low concentration of vesicles in fresh biles (2.0% in TCDC-rich bile and 2.8% in TC-rich bile) suggests that substantial dissolution of vesicles into mixed micelles had occurred before bile collection and experimental observations (10). During equilibration, vesicle dissolution proceeded to completion in TCDCrich and TC-rich biles leaving only mixed micelles and

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trace quantities of protein/glycoprotein aggregates. Unlike rat bile, which contains a mixture of hydrophobic and hydrophilic bile salts (lo), the present experimental design produced prairie dog biles highly enriched in TCDC and TC, which allowed us to attribute the time-dependent dissolution of biliary lipids solely to the hydrophobic nature of the secreted bile salt species. Immediate analysis of TUDC-rich bile revealed the presence of a much larger concentration of vesicles (16%) than initially observed in either TCDC-rich or TC-rich biles suggesting a slower vesicle dissolution rate by TUDC before experimental collection. We interpret this result on the basis of two phenomena that have been established in model systems but not heretofore observed in native biles. First, micellar dissolution rates of model membranes vary directly in proportion to bile salt hydrophobicity and are therefore slow with TUDC (27, 30). Second, the higher cholesterol/ lecithin molar ratios in TUDC-rich bile (Table 1 and Fig. 4C) may also contribute to the cholesterol-rich unilamellar vesicles being more resistant to dissolution by micellar bile salts (11). Because the relative lipid compositions of TUDC-rich bile plotted outside the micellar zone of the phase diagram in Fig. 5C, mixed micelles, vesicles, multilamellar liquid crystals, and/or solid cholesterol monohydrate crystals might be expected to coexist in these biles at equilibrium (4, 6, 8, 16). After 24 h equilibration, the TUDC-rich bile contained mixed micelles, vesicles, and multilamellar liquid crystals but not solid cholesterol monohydrate crystals. Because the liquid crystalline phase could not be harvested in total without significant contamination from the micellar phase, the overall distribution of cholesterol among multilamellar liquid crystals, vesicles, and mixed micelles could not be quantified reliably. However, this distribution was easily estimated. In the TUDC-rich bile (Fig. X), the micellar phase limit plotted at 4 mol/lOO mol cholesterol at a TUDC/lecithin molar ratio = 2.4. The experimentally determined cholesterol content was 6 mol/lOO mol, and therefore ~67% of total cholesterol was solubilized in mixed micelles. Because unilamellar vesicles separated by gel filtration contained ~1% of cholesterol (Fig. 7F), multilamellar liquid crystals must have contained the remaining -33% of cholesterol. Because of the presence of TUDC micelles that solubilized lecithin in excess of cholesterol, the liquid crystalline phase was enriched with cholesterol (cholesterol/ lecithin ratio = 1.0) compared with secreted vesicles (cholesterol/lecithin ratio = 0.41 t 0.16, Table 1; 11). Although multilamellar liquid crystals have been observed in TUDC-rich model and native biles in the presence of excess cholesterol or cholesterol gallstones, this is the first demonstration of the mechanism by which these structures evolve in native biles. Furthermore, our determination of the composition of these multilamellar liquid crystals provides a basis for explaining the absence of solid cholesterol monohydrate crystals in equilibrated TUDC-rich biles: liquid crystals containing cholesterol/ lecithin ratios ~1.0 are stable at equilibrium and coexist with mixed micelles in a two-phase region of the TUDC-

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egg yolk lecithin-cholesterol phase diagram that is devoid of cholesterol crystals (4). Immediate analysis after collection of TUC-rich bile revealed only vesicles. Whereas QLS fails to detect micelles in the presence of relatively high concentrations of vesicles (14), it is unlikely that micelles were present in fresh TUC-rich bile, because the total biliary TUC concentration was only 8.0 mM, a TUC concentration that is less than its reported critical micellar concentration (28) and that fails to solubilize either lecithin or cholesterol in model systems (Fig. 50). As demonstrated by ultracentrifugation and gel filtration, equilibrated TUC-rich bile contained only multilamellar liquid crystals, a phenomenon not previously observed by us or by others. The slightly lower cholesterol/lecithin ratio of liquid crystals in TUC-rich bile (0.8) compared with TUDC-rich bile (1.0) was possibly due in part to the absence of a mixed micellar phase in TUC-rich bile. The complete absence of mixed micelles in TUC-rich bile resulted in a liquid crystalline phase with a cholesterol/lecithin molar ratio of identical value to the cholesterol/lecithin molar ratio of the unfractionated bile (0.67 t 0.15, Table 1). The reason for the absence of cholesterol monohydrate crystals in the TUC-rich bile after 24 h equilibration is due to the stability of the liquid crystalline phase for phase equilibria reasons as discussed above. Taken together, our results support the hypothesis that the coupling of biliary lecithin and cholesterol secretion rates to bile salt secretion rates has, in part, a physicalchemical basis. Based on the presence of vesicles in all biles analyzed immediately after collection, it appears that vesicles provide the common mechanism of biliary lipid secretion despite wide variations in bile salt hydrophobicity. Our results do not exclude the possibility of both a mixed micellar and vesicular pathway for biliary lipid secretion. If this were the case, our data suggest that hydrophilic bile salts would stimulate vesicular secretion of biliary lecithin and cholesterol, whereas more hydrophobic bile salts would promote principally micellar secretion. This possibility is rendered unlikely by observations elsewhere in rats (15) that, at high bile salt secretion rates, bile salts of varied hydrophobicities are cotransported with lecithin and cholesterol into bile by a microtubular-dependent pathway. Therefore, bile salt hydrophobicity influences both absolute biliary lecithin and cholesterol secretion rates and the ratios of cholesterol and lecithin in vesicles destined for bile. After secretion, bile salt hydrophobicity clearly dictates the aggregative behavior of biliary lipids in bile. Rapid dissolution of secreted vesicles in cholesterol unsaturated biles containing more hydrophobic bile salts suggests that mixed micelles are likely to be formed in the biliary tree. Clearly, micelles provide the dominant means of lecithin and cholesterol transport in the biliary tree and gallbladder in the prairie dog and other species with cholesterol unsaturated biles (12). On the other hand, in native biles enriched with more hydrophilic bile salts either naturally (2 1) or pharmacologically (9)) vesicles and liquid crystals may solubilize some or all of biliary lecithin and cholesterol. Hence, mixed micelles, unilamellar vesicles, and macroscopic liquid crystals can all serve independently or

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together as important natural lipids in the biliary tree.

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vehicles for transporting

The authors thank Dr. Joan Staggers for expertise with analysis of the physical states of biliary lipids and Heideh Ahari for superb technical assistance. This study was supported in part by National Institutes of Health Grants DK-36588, AM-34854, and GM-07258. D. E. Cohen was supported by National Institute of General Medical Science Award 5T32GM-07753-08. Present address of L. Leighton: Suite 545, 2001 Peachtree Rd. NE, Atlanta, GA 30309. Address for reprint requests: M. Carey, Dept. of Medicine, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115. Received

9 December

1991; accepted

in final

form

6 April

1992.

AND

15.

16.

17.

18. 19.

REFERENCES 1. Armstrong, M. J., and M. C. Carey. The hydrophobic-hydrophilic balance of bile salts. Inverse correlation between reversephase high performance liquid chromatographic mobilities and micellar cholesterol-solubilizing capacities. J. Lipid Res. 23: 7080, 1982. 2. Bevington, P. R. Data Reduction and Error Analysis for the Physical Sciences. New York: McGraw-Hill, 1969. 3. Bilhartz, L. E., and J. M. Dietschy. Bile salt hydrophobicity influences cholesterol recruitment from rat liver in vivo when cholesterol synthesis and uptake are constant. Gastroenterology 95: 771-779, 1988. M. C. Lipid solubilization in bile. In: BiZe Acids in HeaZth 4. Carey, and Disease, edited by T. C. Northfield, R. P. Jazrawi, and P. L. Zentler-Munro. Dordrecht, the Netherlands: Kluwer Academic Press, 1988, p. 61-82. M. C., and M. J. Cahalane. Enterohepatic circulation. 5. Carey, In: The Liver: Biology and Pathobiology, edited by I. M. Arias, W. B. Jakoby, H. Popper, D. Schachter, and D. A. Shafritz. New York: Raven, 1988, p. 573-616. M. C., and D. E. Cohen. Biliary transport of cholesterol 6. Carey, in vesicles, micelles and liquid crystals. In: BiZe Acids and the Liver, edited by G. Paumgartner, A. Stiehl, and W. Gerok. Lancaster, UK: MTP Press, 1987, p. 287-300. M. C., J.-C. Montet, M. C. Phillips, M. A. Arm7. Carey, strong, and N. A. Mazer. 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 20: 3637-3648, 1981. 8. Carey, M. C., and D. M. Small. The physical chemistry of cholesterol solubility in bile. Relationship to gallstone formation and dissolution in man. J. Clin. Invest. 61: 998-1026, 1978. 9. Carulli, N., P. Loria, M. Bertolotti, M. Ponz de Leon, D. Menozzi, G.Medici, and I. Piccagli. Effects of acute changes of bile acid pool composition on biliary lipid secretion. J. CZin. Invest. 74: 614-624, 1984. D. E., M. Angelico, and M. C. Carey. Quasielastic 10. Cohen, light scattering evidence for vesicular secretion of biliary lipids. Am. J. Physiol. 257 (Gastrointest. Liver Physiol. 20): Gl-G8, 1989. 11. Cohen, D. E., M. Angelico, and M. C. Carey. Structural alterations in lecithin-cholesterol vesicles following interactions with monomeric and micellar bile salts: physical-chemical basis for subselection of biliary lecithin species and aggregative states of biliary lipids during bile formation. J. Lipid Res. 31: 55-70, 1990. 12. Cohen, D. E., and M. C. Carey. Physical chemistry of lipids during bile formation. Hepatology 12: 143S-148S, 1990. 13. Cohen, D. E., and M. C. Carey. Rapid (1 hour) high-performance gel filtration chromatography resolves coexisting simple micelles, mixed micelles and vesicles in bile. J. Lipid Res. 31: 2103-2112, 1990. D. E., M. R. Fisch, and M. C. Carey. Principles of 14. Cohen, laser light-scattering spectroscopy: applications to the physicochemical study of model and native biles. Hepatology 12: 113S122s, 1990.

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Crawford, J. M., C. A. Berken, and J. L. Gollan. Role of the hepatocyte microtubular system in the excretion of bile salts and biliary lipid: implications for intracellular vesicular transport. J. Lipid Res. 29: 144-156, 1988. Donovan, J. M., N. Timofeyeva, and M. C. Carey. Cholesterol monohydrate (ChM) and liquid crystal formation in model biles: effects of bile salt (BS) hydrophobicity and phosphatidylcholine (PC) content (Abstract). Hepatology 10: 598, 1989. Donovan, J. M., N. Timofeyeva, and M. C. Carey. Influence of total lipid concentration, bile salt: lecithin ratio, and cholesterol content on inter-mixed micellar/vesicular (non-lecithin-associated) bile salt concentrations in model bile. J. Lipid Res. 32: 15011512, 1991. Fisher, R. A. The Design of Experiment. Edinburgh, UK: Oliver and Boyd, 1949. Fromm, H., P. Amin, H. Klein, and I. Kupke. Use of a simple enzymatic assay for cholesterol analysis in human bile. J. Lipid Res. 21: 259-261, 1980. Gurantz, D., and A. F. Hofmann. Influence of bile acid structure on bile flow and biliary lipid secretion in the hamster. Am. J. Physiol. 247 (Gastrointest. Liver Physiol. 10): G736-G748, 1984. Hagey, L. R., D. C. Crombie, H. Igimi, M. C. Carey, E. Espinosa, and A. F. Hofmann. Ursodeoxycholic acid in bears: occurrence, site of formation, and biological significance (Abstract). Hepatology 14: 146A, 1991. Heuman, D. M. Quantitative estimation of the hydrophilic-hydrophobic balance of mixed bile salt solutions. J. Lipid Res. 30: 719-730, 1989. Johansen, G., and R. Lumry. Statistical analysis of enzymatic steady-state rate data. Cornpt. Rend. Trav. Lab. Carlsberg 32: 185214, 1961. Mazer, N. A., and M. C. Carey. Quasielastic light scattering studies of aqueous biliary lipid systems. Cholesterol solubilization and precipitation in model bile solutions. Biochemistry 22: 426442, 1983. Mazer, N. A., and M. C. Carey. Mathematical model of biliary lipid secretion: a quantitative analysis of physiological and biochemical data from man and other species. J. Lipid Res. 25: 932953, 1984. Poupon, R., R. Poupon, M. L. Grosdemouge, M. Dumont, and S. Erlinger. Influence of bile acids upon biliary cholesterol and phospholipid secretion in the dog. Eur. J. CZin Invest. 6: 279-284, 1976. Rajagopalan, N., and S. Lindenbaum. Kinetics and thermodynamics of the formation of mixed micelles of egg phosphatidylcholine and bile salts. J. Lipid Res. 25: 135-147, 1984. Roda, A., A. F. Hofmann, and K. J. Mysels. The influence of bile salt structure on self-association in aqueous solutions. J. BioZ. Chem. 258: 6362-6370, 1983. Roslyn, J., J. E. Thompson, and L. DenBesten. Anesthesia for prairie dogs. Lab. Anim. Sci. 29: 542-544, 1979. Salvioli, G., and M. C. Carey. A novel in vitro perfusion system to study membrane dissolution by bile salts: different effects of taurochenodeoxycholate and tauroursodeoxycholate on lipid secretion and membrane resistance (Abstract). Gastroenterology 82: 1168A, 1982. Smallwood, R. A., and N. E. Hoffman. Bile acid structure and biliary secretion of cholesterol and phospholipid in the cat. Gastroenterology 71: 1064-1066, 1976. Staggers, J. E., 0. Hernell, R. J. Stafford, and M. C. Carey. Physical-chemical behavior of dietary and biliary lipids during intestinal digestion and absorption. I. Phase behavior and aggregation states of model lipid systems patterned after aqueous duodenal contents of healthy adult human beings. Biochemistry 29: 2028-2040, 1990. Ulloa, N., J. Garrido, and F. Nervi. Ultracentrifugal isolation of vesicular carriers of biliary cholesterol in native human and rat bile. Hepatology 7: 235-244, 1987. Zar, J. H. BiostatisticaZ Analysis. Englewood Cliffs, NJ: PrenticeHall, 1984.

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