Influence of Phosphatidylcholine and Calcium on Self-Association and

Oct 27, 2015 - ionized UCB and other “calcium-sensitive anions” from bile6,14 and possibly also in cholesterol gallstone formation.6 Therefore, we...
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Influence of Phosphatidylcholine and Calcium on Self-Association and Bile Salt Mixed Micellar Binding of the Natural Bile Pigment, Bilirubin Ditaurate Michael W. Neubrand† and Martin C. Carey* Department of Medicine, Harvard Medical School, and Division of Gastroenterology, Brigham and Women’s Hospital and Harvard Digestive Disease Center, Boston, Massachusetts 02115, United States

Thomas M. Laue Department of Biochemistry, University of New Hampshire, Durham, New Hampshire 03824, United States ABSTRACT: Recently [Neubrand, M. W., et al. (2015) Biochemistry 54, 1542−1557], we determined a concentrationdependent monomer−dimer−tetramer equilibrium in aqueous bilirubin ditaurate (BDT) solutions and explored the nature of high-affinity binding of BDT monomers with monomers and micelles of the common taurine-conjugated bile salts (BS). We now investigate, employing complementary physicochemical methods, including fluorescence emission spectrophotometry and quasi-elastic light scattering spectroscopy, the influence of phosphatidylcholine (PC), the predominant phospholipid of bile and calcium, the major divalent biliary cation, on these selfinteractions and heterointeractions. We have used short-chain, lyso and long-chain PC species as models and contrasted our results with those of parallel studies employing unconjugated bilirubin (UCB) as the fully charged dianion. Both bile pigments interacted with the zwitterionic headgroup of short-chain lecithins, forming water-soluble (BDT) and insoluble ion-pair complexes (UCB), respectively. Upon micelle formation, BDT monomers apparently remained at the headgroup mantle of short-chain PCs, but the ion pairs with UCB became internalized within the micelle’s hydrophobic core. BDT interacted with the headgroups of unilamellar egg yolk (EY) PC vesicles; however, with the simultaneous addition of CaCl2, a reversible aggregation took place, but not vesicle fusion. With mixed EYPC/BS micelles, BDT became bound to the hydrophilic surface (as with simple BS micelles), and in turn, both BDT and BS bound calcium, but not other divalent cations. The calcium complexation of BDT and BS was enhanced strongly with increases in micellar EYPC, suggesting calcium-mediated cross-bridging of hydrophilic headgroups at the micelle’s surface. Therefore, the physicochemical binding of BDT to BS in an artificial bile medium is influenced not only by BS species and concentration but also by long-chain PCs and calcium ions that exert a specific rather than a counterion effect. This work should serve as a physicochemical template for studies with other conjugated bilirubins, including bilirubin diglucuronoside (BDG), the principal bilirubin conjugate (cBR) in human bile.

W

e recently1 investigated the self-association of bilirubin ditaurate (BDT) in aqueous systems, and its interactions with bile salt (BS) monomers and micelles, the principle lipids of bile. BDT is a commerically available, stable analogue of bilirubin diglucuronoside (BDG),2 and we studied it as a potential surrogate compound for BDG, the most abundant conjugated bilirubin (cBR) in human bile.3 BDT is also a natural bile pigment occurring as the only cBR in the Japanese yellowtail (Seriola quinqueradiata) and other marine fish of the genus Seriola.4 Our earlier studies revealed a monomer−dimer equilibrium at low BDT concentrations and ionic strengths and a dimer−tetramer equilibrium at higher BDT concentrations and ionic strengths.1 BDT monomers become tightly bound to both BS monomers and BS micelles, apparently via polar π orbital−H bonding forces, which, in turn, © 2015 American Chemical Society

shifts the equilibrium toward monomers and prevente BDT self-aggregation.1 These results are used herein as a frame of reference for further studies of the interactions of BDT with BS in the presence of the two other major solute components of bile, long-chain phospatidylcholines (PCs, lecithin) and calcium, of particular relevance to biliary lipid biophysical chemistry.5−7 Long-chain PCs, principally palmitoyl-linoleoyl and palmitoyl-oleoyl molecular species,8 are after BS, the most abundant lipids in bile, and form mixed micelles and unilamellar vesicles in the presence of BS.9,10 However, with the lipid proportions, including cholesterol, found in human bile, mixed Received: August 5, 2015 Revised: October 27, 2015 Published: October 27, 2015 6783

DOI: 10.1021/acs.biochem.5b00874 Biochemistry 2015, 54, 6783−6795

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Biochemistry

Sodium salts of the hydroxyl-substituted, 5β-cholanoyltaurates, taurocholic acid (3α, 7α, 12α OH, NaTC), and taurodeoxycholic acid (3α, 12α, NATDC) were purchased from Calbiochem (San Diego, CA) and Sigma (St. Louis, MO). Purified NaTC23 and NaTDC as received were at least 96−99% pure by TLC and HPLC.1 Both bile salts were exhaustively purified further of both free and conjugated congeners and fluorescent impurities (chemical nature uncertain), by an activated charcoal method as summarized previously.1 Synthetic dihexanoyl (C6)-PC and diheptanoyl (C7)-PC, sn1-palmitoyl (C16:0) LPC (Avanti Polar Lipids, Birmingham, AL), and EYPC (Lipid Products, Redhill, Surrey, U.K.) were all at least 99% free of lipid impurities by TLC (200 μg applications). For studies with the calcium-specific electrode, all BS and EYPC were shown to be free of measurable calcium by atomic absorption spectroscopy (model 2280, PerkinElmer, Norwalk, CT). Organic solvents and other chemicals were HPLC and/or ACS reagent grade (Fisher Scientific, Pittsburgh, PA). NaCl was roasted at 600 °C to oxidize and remove organic impurities. NaCl, anhydrous CaCl2, MgCl2, MnCl2, and the tetrasodium salt of ethylenediaminetetraacetic acid (Na4EDTA) (Fisher Scientific or Sigma) were tested as 1% (w/v) solutions by both UV−vis absorbance and fluorescence emission spectrophotometry and found to be free of chromophoric and fluorescent impurities. Ar (Yankee Oxygen, Cambridge, MA) was 99.9% pure, and water was filtered, deionized, and glass-distilled (Corning Automatic System, Corning Glass, Corning, NY). All glassware was sequentially washed (24−48 h) in 2 M KOH dissolved in ethanol and water [1:1 (v/v)] and 1 M HNO3, rinsed in running distilled and deionized water, wrapped in aluminum foil, and dried in an oven. Methods. The conditions under which the experiments were performed were essentially identical to those described previously.1,24 Precautions to avoid lipid and lipopigment degradation were rigorous: water was boiled to remove O2 and CO2 and cooled under Ar. All stock solutions were bubbled with Ar under subdued light throughout the experiments. Under these conditions, the samples remained stable for at least 3 h, and within this time frame, all spectra were fully reproducible. Experiments were performed at room temperature (23 + 1 °C) except measurements with the calcium electrode, which were performed at 37 °C, and experiments with LPC, which were performed at 40 °C because of the high Kraft point (critical micellar temperature) of this saturated monoacyl PC.21 Small unilamellar EYPC vesicles (SUVs) were prepared by the method of Barenholz and colleagues.25 Appropriate lipid concentrations were dried from methanol and chloroform [2:1 (v/v)], hydrated, and hand-dispersed in purified water and sonicated with a Ti probe. Large unilamellar and multilamellar vesicles as well as Ti particles were removed by ultracentrifugation at 160,000g for 15 h. The mean hydrodynamic radius (R̅ h) of the vesicles as determined by quasi-elastic light scattering spectroscopy (QLS) employing “cumulants” analysis26,27 was 120 Å ± 10%, and by Sepharose 4B gel permeation chromatography,28 each sample gave a homogeneous symmetric peak indicating a single vesicle size distribution. Aqueous diC6-PC and diC7-PC as well as LPC stock solutions were prepared by precipitation from MeOH with subsequent drying under reduced pressure to constant weight followed by addition of the aqueous solvent to give gram per deciliter solutions. Mixed micellar solutions of varying BS and EYPC compositions

BS/PC/cholesterol and simple (BS/cholesterol) micelles coexist with a vesicular phase containing PC and cholesterol.10 Although simple and mixed micelles plus vesicles are responsible for solubilization of large quantities of unesterified cholesterol in human bile,10,11 they are also the principle biliary solubilizers of small amounts of unconjugated bilirubin (UCB) in healthy humans.12 Because calcium is the major divalent biliary cation6 and is bound principally by BS in bile,7,13 its free ions (Ca2+) play crucial roles in the precipitation of partially ionized UCB and other “calcium-sensitive anions” from bile6,14 and possibly also in cholesterol gallstone formation.6 Therefore, we also studied the effects of CaCl2 on the interactions of BDT with simple BS and mixed BS/long-chain (egg yolk, EY) PC micelles and EYPC vesicles individually because they coexist under physiological conditions.10,15 We have also investigated the interactions of BDT and dianionic UCB with micelleforming short-chain PCs, as well as lysoPC (LPC), which have been extensively characterized by themselves in aqueous media.16−21 Both our UV−visible (UV−vis) and fluorescence spectrophotometric results suggest that BDT monomers in soluble ion-pair complexes with short- and single-chain PC monomers partition into a hydrophilic environment of short-chain PC and LPC micelles. In contrast, short-chain PC micelles solubilize in their hydrophobic cores water-insoluble ion-pair complexes of dianionic UCB and PC. BDT becomes bound to a hydrophilic environment of BS/EYPC micelles, and mostly pure BS binding was inferred from a lack of an appreciable effect of EYPC content. Added calcium enhanced binding of BDT to mixed micelles in proportion to increases in EYPC levels, suggesting that the divalent cation changed the configuration of BDT on BS/EYPC micelles partly in favor of zwitterionic PC headgroup binding. The combination of both BDT and calcium led to reversible aggregation but not fusion of small unilamellar vesicles (SUVs) of EYPC, and BDT, apparently as the calcium salt was internalized into the vesicle bilayer. This interaction was not induced by similar concentrations of other biliaryrelevant divalent cations, suggesting that it was not a simple counterion effect but was calcium-specific. Although occurring physiologically in nature, BDT is not present in human bile. Therefore, the question of whether the physicochemical properties of this major bilirubin conjugate of human bile are anticipated by the present studies with BDT remains to be investigated.

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EXPERIMENTAL PROCEDURES Materials. Crystalline BDT and UCB were obtained from Frontier Scientific, formerly Porphyrin Products (Logan, UT). Gram quantities of each bile pigment were especially purified by recrystallization and were supplied in divided portions in ampules composed of actinic glass under a blanket of Ar. The chemical purity determined by thin layer chromatography (TLC),22 high-performance liquid chromatography (HPLC), and UV−vis absorption spectrophotometry was ≥99% as described in detail in our earlier paper.1 Specifically, this synthetic BDT contained no lipid impurities and no monoconjugated bile pigments, UCB, or biliverdins; 75% consisted of the natural IXα isomer with 15 and 10% IIIα and XIIIα constitutional isomers, respectively.1 UCB was free of contaminating biliary lipids, and conjugated bile pigments, and was principally the IXa isomer, and constitutional isomeric impurities accounted for 2% (IIIα) and 4% (XIIIIα). 6784

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particles provided the mean hydrodynamic radius (R̅ h) using the Stokes−Einstein relationship.

were prepared by coprecipitation from CHCl3 and MeOH (2:1 to 1:1 by volume) as described previously.5 For calcium ion activity by specific electrode measurements, the dried lipid films were reconstituted in aqueous 8 mM CaCl2 and 126 mM NaCl (ionic strength of 0.15) as detailed elsewhere.7 According to the intended spectrophotometric experiments, stock BDT solutions (pH 6−7) were combined in a 1:1 or 1:2 (v/v) ratio with stock solutions of the other lipids with and without appropriate concentrations of neutral electrolytes, all prepared gravimetrically to give the desired concentrations.1 Stock solutions of dianionic UCB were prepared gravimetrically by weighing the appropriate amount of UCB, adding a few drops of 1 M NaOH, and dissolving the solute to the desired solution volume with 0.01 M carbonate-bicarbonate buffer at pH 10. Serial dilutions were then conducted with the BDT and UCB/electrolyte solutions to vary the lipid concentrations at constant pH and a neutral electrolyte concentration. For all spectrophotometric experiments, BDT concentrations of 20−100 μM were studied. By this means, absorbance values were maintained between 0.05 and 1. The tubes were wrapped in aluminum foil and capped under a blanket of Ar. All experiments were performed in the dark with the aid of safety lights. Over an experimental period of 1−3 h, absorbance values at 410−490 nm remained constant. UV−vis absorption spectra were recorded on a Cary 118C spectrophotometer (Varian Associates, Palo Alto, CA). To further keep absorbance values between 0.05 and 1, matched glass cuvettes with path lengths of 1 and 2 mm were employed. Otherwise, all UV−vis absorbance conditions were identical to those outlined previously.1,24 The slit width was autocontrolled, and the temperature (see the figure legends) was regulated by thermostatically calibrated water jackets connected to an automated heat exchanger (K-2/RD, Lauda, Königshafen, Germany). Fluorescence emission spectra were recorded on a PerkinElmer (Norwalk, CT) fluorimeter. Slit widths for excitation and emission, the path length, and other experimental conditions were identical to those described previously.1 Accurate corrections for the “inner-filter effects” caused by 40 μM BDT were performed according to the method of Knudsen and associates29 as summarized earlier.1 Correction factors were accurate to within 1% because UV−vis absorbances were kept at 0.999) was constructed. From stock solutions, standard CaCl2 concentrations (0.1−20 mM) were mixed with NaCl solutions as needed, to achieve a total ionic strength of 0.15. Unbound calcium in a BDT solution was calculated from the calibration curve, with the y-intercept corrected for the 8 mM Ca2+ standard. Calcium binding was calculated from the equation [bound calcium] = 8 mM − [unbound calcium]

(2)

While no attempt was made to determine the actual stoichiometric interactions between Ca and BDT, we calculated apparent formation constants (Kf) according to the formula6

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K f = ([Ca tot] − [Ca 2 +])/[Ca 2 +][BDT])

(3)

RESULTS Interactions of BDT and Dianionic UCB with ShortChain PCs. Figure 1a summarizes UV−vis absorbance spectra for 100 μM BDT with diC6-PC at concentrations (0−58 mM) varying from well below to above its critical micellar concentration (CMC) of 14 mM.16 With increases in diC6PC concentration to the CMC value, the maximal absorbance (Amax = 450 nm) of BDT decreases, a slight bathocromic shift occurs, and a hypsochromic shoulder forms. With diC6-PC concentrations above its CMC, a new Amax becomes established at 415 nm. UV−vis spectra recorded with 100 μM BDT and diC7-PC (data not displayed) show similar behavior except that the transition point to the hypsochromic λmax occurs at PC concentrations of ≈2 mM close to its CMC value.16 Figure 1b shows UV−vis absorbance spectra of 25 μM disodium UCB (pH 10.1) with a range of diC 6 -PC concentrations similar to that in Figure 1a. The CMC of diC6-PC16 at pH 6.9 should be pH-independent because the phosphorylcholine headgroup of PC is zwitterionic between pH 2 and 12.32 In the absence of diC6-PC, the λmax of UCB is 440 nm. In contrast to the BDT/diC6-PC systems (Figure 1a), two striking differences are noted. First, below the CMC, Amax decreases monotonically and the spectra do not form a shoulder. Second, with micellar diC6-PC concentrations, λmax shifts bathochromically and Amax increases appreciably. Figure 2 displays absorbance ratios (A416/A450) of 100 μM BDT as functions of diC6-PC and diC7-PC concentrations. 6785

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Figure 1. (a) Absorption spectra of 100 μM BDT (path length of 1 mm, absorption range of 1) with changing diC6-PC concentrations. Spectra 1−6 correspond to diC6-PC concentrations of 0, 11.6, 14.5, 19.3, 38.5, and 58.8 mM, respectively (23 °C, pH 7.5−8.0). (b) Absorption spectra of 25 μM UCB (path length of 2 mm, absorption range of 0.5, pH 10.1) with changing diC6-PC concentrations. Spectra 1−7 correspond to diC6-PC concentration of 0, 1.8, 3.7, 4.9, 7.5, 15, and 37 mM, respectively. Note the red-shift with micellar concentrations of the short-chain PC (curve 7) and the decreases in UCB absorbance with monomeric PC concentration (curves 1−5).

Figure 3. Absorption spectra of bilirubins in aqueous lysophosphatidylcholine (LPC) systems. (a) Absorption spectra of 20 μM BDT (path length of 10 mm, absorption range of 2) with changing LPC concentrations. Spectra 1−5 correspond to LPC concentrations of 0, 62.5, 125, 250, and 500 μM, respectively, all micellar at 40 °C (pH 7.5−8.0). (b) Absorption spectra of 20 μM UCB (path length of 10 mm, absorption range of 2, pH 10) with changing LPC concentrations. Spectra 1−5 correspond to LPC concentrations of 0, 62.5, 125, 250, and 500 μM, respectively (40 °C).

Amax of UCB shifts to the red and concomitantly a progressive decrease in A440 is accompanied by an increase in A460. A tight isosbestic point is noted (Figure 3b), but in contrast to the UV−vis spectra of BDT/LPC mixtures (Figure 3a), it does not include the spectrum of pure UCB. Calcium Binding by BDT Solutions. Figure 4 displays a plot of bound calcium in millimolar as a function of BDT

Figure 2. Dependence of absorbance ratios (416 nm to 450 nm) of 100 μM BDT with increases in diC6-PC and diC7-PC concentrations. Once the corresponding CMCs, as evaluated by static light scattering spectroscopy, are reached (ref 16), abrupt increases in absorbance ratios occur. T = 23 ± 1 °C, pH H2O (6−7), no added inorganic salt. Figure 4. Calcium binding by BDT as assayed by the calcium-specific electrode. Conditions for triplicate measurements (see Experimental Procedures): 37 °C, 126 mM NaCl, 8 mM CaCl2, μ = 0.15. The curvilinear relationship shows that at the lowest BDT concentration, one calcium ion is bound per two BDT molecules, whereas at the highest BDT concentration, one calcium ion is bound per seven BDT molecules.

With short-chain PC concentrations below their respective CMC values16 (arrows), absorbance ratios show no appreciable change. At the CMC, a sharp increase in absorbance ratios occurs, leveling off at stable values above 8 mM (diC7-PC) and 19 mM (diC6-PC). Figure 3a shows overlapped spectra of 20 μM BDT with increasing LPC concentrations (40 °C, pH 7.5−8.0). All PC concentrations employed (62.5−50 μM) are appreciably higher than the CMC of LPC in H2O.21 Increases in LPC concentration result in decreased absorbances of BDT at 450 nm, a slight shift to the red, and the formation of a hypsochromic shoulder at ≈420 nm. However, in contrast to the short-chain PCs, a tight isosbestic point occurs with BDT/ LPC solutions at 427 nm, indicative of two molecular species in equilibrium. Figure 3b summarizes spectra of 20 μM dianionic UCB (pH 10) for the same range of LPC concentrations (as in Figure 3a). With increases in micellar LPC concentration, the

concentration to a concentration of 50 mM, which is close to the “salting out” limit under the conditions of ionic strength and temperature employed (126 nM NaCl, 8 mM CaCl2, μ = 0.15, and 37 °C). There is a curvilinear increase in bound calcium as functions of increases in BDT concentration in that the molar ratio of calcium bound to BDT decreases from 0.52 at 5 mM BDT to 0.15 at 50 mM BDT. This equates to one calcium ion bound per two BDT molecules at the lowest BDT concentrations to one bound to seven BDT molecules at the highest BDT concentration. Calculated per sulfonate group, Ca 6786

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Figure 6b), very little change occurs in the fluorescence spectra compared with that of BDT alone (curve 1 in each panel). With BS concentrations higher than the CMC values, progressive increases in the relative fluorescence intensities (RFI) occur. At the highest NaTC concentration (50 mM), RFI values at λmax increase by a factor of 2.4 above baseline (Figure 6a) and λmax emission shifts to the blue. At corresponding NaTDC concentrations, RFI values of BDT increase by a factor of 8 over baseline (Figure 6b). In each case, the BS induces increases in RFI values that are 2-fold greater with 50 mM CaCl2 than with 150 mM NaCl.1 Panels a and b of Figure 7 plot all RFI values at BDT’s λmax for a range of NaTC and NaTDC concentrations each at a

binding by BDT is 2.5-fold greater that calcium binding by NaTC7 for otherwise identical conditions, with Kf values ranging from 203 to 280 L/mol. Influence of CaCl2 on BDT Absorption Spectra in Bile Salt Solutions. Figure 5 plots absorbance ratios (Amax/A450) of

Figure 5. Absorbance ratios of 100 μM BDT showing marked increases in micellar solutions of bile salts and then leveling off. Solutions of NaTDC with 50 mM CaCl2 (●), NaTC with 50 mM CaCl2 (◆), NaTDC with 150 mM NaCl (□), and NaTC with 150 mM NaCl (△) at 23 °C.

100 μM BDT in NaTC and NaTDC solutions (0−50 mM) with added 50 mM CaCl2 (filled symbols) or 150 mM NaCl (empty symbols). Compared to a similar plot in H2O1 and with added NaCl, the partitioning of BDT from H2O (A450) to micelles (Amax) with added CaCl2 occurs more abruptly and completely over a narrower BS concentration range. Panels a and b of Figure 6 depict emission spectra of 40 μM BDT with increasing concentrations (0−50 mM) of NaTC and NaTDC, respectively, in the presence of 50 mM CaCl2. With monomeric NaTC (curves 2 and 3 in Figure 6a) or NaTDC (curve 2 in

Figure 7. (a) RFI values of 40 μM BDT in a range of NaTC concentrations (indicated on the abscissa) as a function of varying CaCl2 concentrations (0−50 mM). (b) Corresponding curves for NaTDC in the presence of an identical range of CaCl2 concentrations (pH 7.5−8.0, 23 °C, path lengths of the exciting and emitting light beams of 3 and 10 mm, respectively). The RFI values of BDT with NaTDC are considerably higher than with NaTC, and the change in slope is critical at low NaTDC concentrations.

constant CaCl 2 concentration (0−50 mM CaCl 2 ). At equivalent BS concentrations, all RFI values are appreciably lower in NaTC (Figure 7a) than in NaTDC (Figure 7b) and the increases at the approximate CMC values (1−2 mM for NaTDC and 5−8 mM for NaTC)9,33−35 take place abruptly in the case of NaTDC and progressively in the case of NaTC. These phenomena are most marked in proportion to increases in CaCl2 concentration (Figure 7). RFI values for submicellar BS concentrations (Figure 7) show only small differences between BS species and are totally independent of CaCl2 concentration (amplified data not displayed). These RFI values for BDT in response to added CaCl2 reflect the critical nature of the CMC in the case of NaTDC micellization and the “uncritical” nature of NaTC micellization even with added ionic strength.9,35 Steady state anisotropy measurements were performed as a function of the variation in CaCl2 concentration to elucidate the effects of neutral electrolytes on the relative mobility of BDT in micellar BS solutions (see Experimental Procedures). As displayed in Figure 8, CaCl2 induces marked decreases (15− 20%) in the anisotropy of BDT molecules (i.e., increased

Figure 6. (a) Dependences of relative fluoroscence intensities (RFI) on emission wavelength (nanometers) for 40 μM BDT in the presence of 50 mM CaCl2. Spectra 1−6 correspond to added NaTC concentrations of 0, 2.5, 5, 10, 25, and 50 mM, respectively. (b) Dependences of RFI values on emission wavelength (nanometers) for 40 μM BDT in the presence of 50 mM CaCl2. Spectra 1−7 correspond to added NaTDC concentrations of 0, 1.25, 2.5, 5, 10, 25, and 50 mM, respectively (pH 7.5−8.0, 23 °C, path lengths of the exciting and emitting light beams of 3 and 10 mm, respectively). 6787

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higher RFI value and a lower anisotropy ratio (less ordered system) compared with those of the NaTC system, suggesting greater mobility of BDT molecules in and/or on dihydroxy BS micellar systems with added CaCl2 and with NaCl (Table1). Interactions of BDT with BS/EYPC Micelles. Figure 9a summarizes the overlapped UV−vis spectra of 50 μM BDT in

Figure 8. Influence of increases in CaCl2 concentration on anisotropy ratios, expressed as the percent decrease in anisotropy, of 40 μM BDT in 50 mM micellar solutions of NaTC (●) and NaTDC (○) (23 °C, pH 7.5−8.0). The larger the percent decrease, the more mobile the BDT microenvironment, i.e., NaTDC > NaTC.

mobility) in micellar NaTDC, and the sharpest change occurs between 0 and 10 mM CaCl2. In contrast, only a gradual anisotropy decrease of ≈5% is observed over a similar range of NaTC concentrations (Figure 8). Table 1 lists RFI values and anisotropy ratios for BDT with NaTC and NaTDC as a function of added CaCl2 concentration

Figure 9. UV−vis absorption spectra of 50 μM BDT in 3 g/dL mixed BS/EYPC micellar solutions. (a) Spectra for BDT (1) and three NaTC/EYPC mixtures (2−4), all without CaCl2. (b) Spectra for BDT and three NaTC/EYPC mixtures with 15 mM CaCl2. The numbered spectra correspond consecutively to changing BS:EYPC molar ratios of (1) BDT only (no BS or EYPC), (2) 10:0, (3) 8:2, and (4) 6:4 (path length of 2 mm, absorbance range of 1, pH 7.5−8.0, 23 °C).

Table 1. Influence of Neutral Electrolytes on Mean Hydrodynamic Radii (R̅ h), Relative Fluorescence Intensities (RFI), and Anisotropy Ratios of NaTDC/BDT and NaTC/ BDT Systemsa

50 mM NaTDC and 50 μM BDT with CaCl2 with CaCl2 with CaCl2 with CaCl2 with CaCl2 with NaCl 50 mM NaTC and 50 μM BDT with CaCl2 with CaCl2 with CaCl2 with CaCl2 with CaCl2 with NaCl

added ionic strength (mM)

R̅ h (Å)

RFI

anisotropy ratiob

0 5 10 20 50 150

12 12 12 21 34 18

3.1 5.9 9.6 17.0 25.8 12.8

0.274 0.248 0.234 0.229 0.219 0.230

0 5 10 20 50 150

11 9 12 12 12 16

2.6 3.6 4.1 5.3 6.8 5.5

0.300 0.295 0.291 0.286 0.283 0.291

NaTC/EYPC micellar solutions (3 g/dL) in H2O as a function of increasing molar ratios of EYPC to BS. Compared with BDT alone (curve 1 in Figure 9a), the spectra show a hypsochromic Amax when the EYPC:BS ratio of the mixed micelles is increased (curves 2−4 in Figure 9a) as well as decreased absorbances at both 450 and 420 nm. Figure 9b displays the results for an identical experiment conducted with added 15 mM CaCl2. In the presence of CaCl2, there is broadening of the absorbance spectrum for BDT alone (curve 1 in Figure 9b), most likely caused by a counterion (Ca2+)-induced aggregation of BDT monomers (see ref 1). With added CaCl2, increased absorbance at 420 nm in the EYPC/BC micellar system is accompanied by sharp decreases in Amax at 450 nm, and these hypsochromic shifts are more pronounced with Ca2+ (Figure 9b) than without Ca2+ (Figure 9a). Panels a and b of Figure 10 display fluorescence emission spectra obtained with 40 μM BDT for otherwise identical conditions as in panels a and b of Figure 9. The RFI values of BDT without CaCl2 decrease with increasing micellar EYPC:BS molar ratios (Figure 10a), but in the presence of 10 mM CaCl2, the RFI values show the opposite trend, increasing with augmented EYPC:BS molar ratios (Figure 10b). In the absence of CaCl2, λmax emission is identical for all PC contents in the mixed micelles (Figure 10a), whereas in the presence of CaCl2 (Figure 10b), a bathochronic shift in λmax emission occurs. Interactions of BDT with SUVs of EYPC. Figure 11 summarizes UV−vis spectra of 50 μM BDT without (curves 1) and with (curves 2) SUVs of EYPC (1 mg/mL) under the following conditions: (a) pure H2O, (b) with 150 mM NaCl, (c) with 20 mM MgCl2, (d) with 50 mM MnCl2, and (e) with 10 mM CaCl2. The experimental conditions were chosen so that the BDT monomer−dimer equilibrium was approximately the same for all electrolyte concentrations with the exception of

All recorded at room temperature (23 ± 1 °C). bFor ease of comprehension, the smaller the number, the less ordered the fluorophore. a

and compares the values with those in 150 mM NaCl. Because micellar size may account for the BS species differences in RFI and anisotropy values for BDT, R̅ h values of NaTC and NaTDC without BDT by QLS are also listed. CaCl2 added to a concentration of 50 mM triples the size of NaTDC micelles compared with NaTC micelles (R̅ h values of 34 and 12 Å, respectively). In the NaTDC/BDT system, increments in micellar sizes with added calcium correlate positively with increases in RFI values and negatively correlate with decreases in anisotropy ratios, indicating a less ordered system at the highest calcium contents. For comparable micellar sizes [e.g., R̅ h = 12 Å (Table 1)], the NaTDC system displays an appreciably 6788

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H2O alone and with added CaCl2. Besides the minimal reduction in absorbances from SUV-induced light scattering, the spectra of BDT in H2O or with NaCl (Figure 11a,b) change neither their shape nor λmax. The BDT spectra with MgCl2 and MnCl2 (Figure 11c,d) show a slight hypsochromic shift in Amax compared with that with NaCl (Figure 11b), and the presence of SUVs decreases absorbance values and slightly increases the hypsochromic shifts. In contrast, addition of 10 mM CaCl2 (Figure 11e) results in a profound influence on the BDT spectra of the SUV system. The absorbance at 450 nm decreases and increases markedly at 423 nm, resulting in a hypsochromic λmax with an absorbance value that exceeds the original Amax at 450 nm. Because the results in Figure 11 strongly suggest that BDT was forced into a different environment, presumably SUV binding, by the presence of calcium but not by other divalent (or monovalent) cations, we followed this observation with a systematic examination of the effects of calcium on BDT−SUV interactions. Figure 12 depicts overlapped spectra of 50 μM BDT with 1 mg/mL SUVs of EYPC as a function of systematic increases (0−30 mM) in CaCl2 concentration. Occurring at the lowest CaCl2 concentration (1 mM, curve 2) is a marked decrease in absorbance at 450 nm and a corresponding increase in absorbance at 420 nm. With every increment in CaCl2 concentration, these spectral changes become more pronounced. The overlaid spectra (Figure 12) display a very tight isosbestic point at 432 nm, indicative of only two BDT molecular species in equilibrium.

Figure 10. Fluoroscence emission spectra of 40 μM BDT alone and in the presence of 3 g/dL mixed micellar solutions of NaTC and EYPC (a) without CaCl2 and (b) with 10 mM CaCl2. Spectra 1−4 correspond to the NaTC:EYPC molar ratios of 10:0, 8:2, 7:3, and 6:4, respectively. Note that in the presence of CaCl2, RFI values (b) increase with increasing EYPC content (less mobile), whereas in the absence of CaCl2 (a), RFI values decrease with increasing EYPC content (more mobile).

Figure 11. UV−vis absorption spectra of 50 μM BDT alone (spectra 1) and with small unilamellar vesicles (SUVs) of EYPC (1 mg/mL) (spectra 2). Both sets of spectra were obtained under the following conditions: (a) in water with no added neutral electrolytes, (b) with NaCl (150 mM), (c) with MgCl2 (50 mM), (d) with MnCl2 (50 mM), and (e) with CaCl2 (10 mM). Note the slight blue-shift with all added electrolytes compared with water but a more marked counterion effect with 10 mM CaCl2. 6789

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mM CaCl2 alone and 10 mM CaCl2 with 150, 300, and 600 mM NaCl, respectively. The spectra of BDT/SUV systems with CaCl2 exhibit strong increases in RFI values in proportion to CaCl2 concentration with parallel shifts of the λmax (emission) to the red. When NaCl (150−600 mM) was added in addition to 10 mM CaCl2 (curves 4−6 in Figure 13b), a decrease in RFI values, but no reversal in spectral shift, occurs. Tables 2 and 3 list the R̅ h values of SUVs in the presence of BDT, CaCl2, or both. When studied over a period of several Table 2. Influence of Calcium Chloride on SUV Sizes without and with 50 μM BDTa

Figure 12. Absorption spectra of 50 μM BDT (path length of 2 mm, absorption range of 1) with SUVs of EYPC (1 mg/mL) with increasing CaCl2 concentrations. Spectra 1−9 correspond to CaCl2 concentrations of 0, 1, 2, 3.5, 5, 8, 14, 21, and 30 mM, respectively (pH 7.5−8.0, 23 °C).

a

[CaCl2] (mM)

R̅ h of SUVs (Å)

R̅ h of SUVs with BDT (Å)

0 10 20 30

110 115 115 115

110 130 150 165

Data collected a room temperature (23 ± 1 °C).

Table 3. Influence of BDT Concentration on SUV Sizes with and without 5 mM Calcium Chloridea

To explore this phenomenon further, fluorescence spectra of BDT/SUV solutions containing CaCl2 show marked differences compared to BDT/SUV spectra with other mono- and divalent electrolytes. Figure 13a displays fluorescence spectra of 40 μM BDT (curve 1) with 1 mg/mL SUVs of EYPC (curve 2) and increasing (3−20 mM) CaCl2 concentrations (curves 3−6 in Figure 13a). Figure 13b shows a corresponding set of spectra, except that curves 3−6 contain a combination of 10

a

[BDT] (μM)

R̅ h of SUVs (Å)

R̅ h of SUVs with CaCl2 (Å)

0 62 125 250

110 113 115 115

115 130 140 270

Data collected a room temperature (23 ± 1 °C).

hours by QLS, SUVs do not grow significantly with increasing concentrations of either CaCl2 [0−30 mM (Table 2)] or BDT [0−250 μM (Table 3)]. However, in the presence of both CaCl2 and BDT, SUVs became progressively larger as a function of increases in CaCl2 concentration [110−165 Å (Table 2)] and more especially with increases in BDT concentration [115−270 Å (Table 3)]. To test if this enlargement was caused by fusion or by aggregation of SUVs, 100 mM Na4EDTA was added to CaCl2containing SUV/BDT solutions. Figure 14a plots overlapped UV−vis spectra and Figure 14b fluorescence emission spectra of BDT with SUVs (curves 1) with either Na4EDTA (curves 2), CaCl2 (curves 3), or both (curves 4). Addition of 100 mM Na 4 EDTA to the pure BDT/SUV system shifts λ max hypsochromically, demonstrating an appropriate Na+ counterion effect (equivalent to 400 mM NaCl) on BDT alone (see ref 1). In Figure 14a, curve 3 shows the expected effect of 10 mM CaCl2 on the BDT/SUV spectra (see Figure 11). When Na4EDTA was added to this system, i.e., a CaCl2/BDT/SUV solution, the spectrum (curve 4 in Figure 14a) reverts to that of a BDT/SUV/Na4EDTA solution without CaCl2 (curve 2 in Figure 14a). Analogous observations with the RFIs in the fluorescence emission spectra (Figure 14a) are made. When Na4EDTA is added to the highly flourescent BDT/SUV/CaCl2 system (curve 3 in Figure 14b), the spectrum (curve 4 in Figure 14b) quenches to essentially that of a BDT/SUV/Na4EDTA system (curve 2 in Figure 14b). It is clear that addition of Na4EDTA fully reverses the profound effects of 10 mM CaCl2 on both sets of BDT/SUV spectra.

Figure 13. Fluorescence emission spectra of 40 μM BDT with and without SUVs of EYPC (1 mg/mL) and added electrolytes. In panel a, spectra 1−6 correspond to BDT alone, BDT with SUVs, and BDT with SUVs and 3, 10, 15, and 20 mM CaCl2, respectively. (b) Spectra 1−6 correspond to BDT alone, BDT with SUVs, BDT with SUVs and 10 mM CaCl2, and BDT with SUVs, 10 mM CaCl2, and 150, 300, and 600 mM NaCl, respectively (pH 7.5−8.0, 23 °C, path lengths of exciting and emitting light beams of 3 and 10 mm, respectively).

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DISCUSSION In this study, we have attempted to address a global systematic approach to BDT in artifical bile solutions. As evaluated by us 6790

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first studying micelle-forming short-chain PCs, we found that the extent of BDT’s interaction with PC increased markedly when micellization occurred (Figures 1a and 2). We found that the BDT probe did not perturb the microenvironment appreciably because the observed CMC values with BDT were in perfect agreement (Figure 2) with those in the literature measured noninvasively employing static light scattering, surface tension, and equilibrium sedimentation measurements.16,17 As occurs with most added reporter molecules of opposite charge,41,42 the diminution in CMC values below the noninvasive CMC, most obvious with diC6PC because it exhibited the higher CMC (Figure 2), suggests an ionic interaction between the negatively charged sulfonates of BDT and the positively charged cholines of PC monomers, resulting in a less water-soluble ion-pair BDT−PC complex.42 Above their “true CMCs” (Figures 1 and 2), the hydrophobic ion-pair complex is internalized by solubilization in PC micelles producing the UV−vis spectral shift (Figure 1) and altering the A416/A450 absorbance ratio (Figure 2). It appears from the hypsochromic shift of BDT’s λmax (Figure 1), indicative of a less hydrophobic environment, that the chromophores of BDT lie close to the zwitterionic polar headgroups of the short-chain PC micelles. As inferred from the absence of an isosbestic point in the spectra (e.g., Figure 1), the micellar equilibria of BDT with PC must involve at least three BDT species in distinct molecular environments, presumably BDT monomers, ion pairs of BDT and PC, and ion pairs of BDT and PC internalized in or on micelles. These results suggested that similar interactions should occur between BDT and LPC. However, UV−vis spectra of BDT at micellar LPC concentrations (Figure 3a) revealed the formation of a tight isosbestic point at 426 nm, suggesting an equilibrium of only two BDT species. The difference between LPC and short-chain PCs with BDT is more apparent than real because the very low CMC (∼7 × 10−6 M) of palmitoyl LPC18 would favor minute concentrations of BDT−LPC ion-pair complexes that would escape UV−vis detection. Besides, these ion pairs may have been insoluble and precipitated from solution or adhered to glass because of their hydrophobicity.42 The interactions of dianionic UCB with micelle-forming PCs including LPC further support this interpretation. The progressive hypochromia of UCB below the CMC of diC6PC (Figure 1b) suggests insoluble ion-pair formation and microprecipitation. The appropriate UV−vis absorbance of UCB is restored just above the CMC of diC6-PC, suggesting solubilization of the phase-separated complex. At much higher micellar diC6-PC concentrations (curve 7 in Figure 1b), the absorbance band is split with a bathochromic λmax, indicating that the environment of UCB is distributed between the micellar surface and hydrophobic core. This contention is further supported by the UV−vis spectra of dianionic UCB with LPC micelles (Figure 3b). The progressive bathochromic shift in the UV−vis spectra of UCB with all increases in micellar LPC indicate that UCB interacts preferentially with the hydrophobic interior of the micelles. The isosbestic point is indicative of an equilibrium between aqueous dianionic UCB and micelle-solubilized UCB presumably as ion-pair complexes with LPC. The fact that the isosbestic point did not include the spectrum of pure UCB, i.e., no added LPC (Figure 3b), is indicative of microprecipitation of UCB−LPC ion pair complexes. When the Amax values of all UCB−LPC spectra during microprecipitation are reduced, the curves are shifted hypochromically compared with those for UCB alone in that at

Figure 14. (a) Absorption spectra of 40 μM BDT with SUVs (1 mg/ mL) of EYPC: (1) BDT/SUV, (2) BDT/SUV/100 mM Na4EDTA, (3) BDT/SUV/10 mM CaCl2, and (4) BDT/SUV/10 mM CaCl2/100 mM Na4EDTA (path length of 2 mm, absorbance range of 1). (b) Fluoroscence emission spectra of of 40 μM BDT with SUVs of EYPC: (1) BDT/SUV, (2) BDT/SUV/100 mM Na4EDTA, (3) BDT/SUV/ 10 mM CaCl2, (4) and BDT/SUV/10 mM CaCl2/100 mM Na4EDTA (path lengths of the exciting and emitting light beams of 3 and 10 mm, respectively).

previously,1 we proposed that BDT behaves in solution as an excellent model compound for cBRs in general. Besides, it is natural bile pigment occurring in marine fish of the Seriola genus. We submit here that it has allowed us to gain insights into how bilirubin diglucuronoside might behave in human bile. In the work presented here, we have modeled native bile by studying the interactions of BDT with bile salts (taurine conjugated as in our earlier study1) and the phospholipid phosphatidylcholine and Ca2+, which are typical biliary components of carnivores and omnivores. We have studied BDT in the presence of EYPC, a biliary analogue, and have used model compounds such as short-chain PCs and LPC to understand the impact of the interactions of Ca2+ ions on the binding interactions of BDT and BS in the presence of mixed bile salt/EYPC micelles. Interactions of BDT with Short-Chain PCs and BS/ EYPC Micelles. We infer from earlier studies24,36−39 that the predominant microdomain for binding of UCB IXα to BS is determined by pH, BS species, and BS concentration. Dianionic UCB IXα (at pH 10) at low BS concentrations appears to be hydrophilically attached to the polar face of BS micelles but progressively partitions into the hydrophobic interior of micelles with increasing BS concentrations.24,40 In contrast, the much more hydrophilic BDT was shown by us earlier1 to interact only with the hydrophilic face of BS monomers and micelles, irrespective of BS species or concentration. The hydrophilic association of BDT with BS is reflected in the difficulty in determining CMC values of BS with BDT as a spectroscopic probe.1 In contrast, accurate CMC values of BS micellization have been determined spectrophotometrically with dilute dianionic UCB24 and with bilirubin monoglucuronide (BMG) at pH 7.8,40 as reporter probes. These findings taken together suggest that as bilirubin IXα becomes more hydrophobic (dianionic UCB > BMG > BDT), the BS micellar binding domain changes from a hydrophilic one to a hydrophobic one. The major focus of this work was to obtain information about the influence of PCs and calcium on BS−bile pigment interactions using dianionic UCB and BDT as the two extremes in the hydrophilic−hydrophobic balance of the molecules.39 By 6791

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of BDT molecules by micellar binding. The marked effects of calcium on increasing RFI values (Table 1) in the case of NaTDC, in particular, are associated with micellar growth and a sharp decrease in anisotropy ratios, indicating less ordering of the BDT microenvironment. With NaTC, the RFI values increased slightly (Figure 6) but no appreciable change in the anisotropy ratio occurred (Table 1). From the anisotropy ratios, BDT appeared to be held in a more rigid environment in NaTC micellar systems than NaTDC micelles for identical micellar sizes (Table 1). In summary, these data further support the proposal made previously1 that BDT and BS interact via strong π−H bonding to the α-hydrophilic faces of BS molecules and that the rigidity of the interaction depends upon the number of α-oriented OH groups and not upon micellar size, nor as inferred from this work the degree of calcium binding to BDT or BS. A phenomenon that is not as easily explained is why added CaCl2 leads to further increases in the already mobile BDT molecule in NaTDC micelles but has only a minor effect on NaTC/BDT micellar systems (Table 1). It is unlikely that cross-chelation occurred because this type of bridging should have the opposite effect. It is more likely that the high concentrations of the calcium dication somehow interfered with π−OH interactions on the polar surface of the micelles that allowed a greater flexibility of BDT molecules to rotate on NaTDC micelles or caused more rapid on/off kinetics of BDT in NaTDC solutions compared to the lifetime of the fluorescent excited state.31 CaCl2 induces even more profound interactions in the binding of BDT to BS/EYPC mixed micelles and did so in proportion to the EYPC content, which by itself had increased the level of micellar BDT binding slightly (Figure 9 and ref 39). Calcium ions facilitated partitioning of BDT onto the hydrophilic surface of pure BS micelles (Figure 5) and possibly also through ionic complexation of BDT and EYPC via Ca bridges in mixed micelles (Figure 9). As was noted with short-chain PC micelles (Figures 1 and 2), BDT interacts with zwitterionic headgroups of PC without calcium. The fact that BDT partitions onto the BS/EYPC mixed micelles more readily when the EYPC content is high suggests that the zwitterionic PC headgroup is involved in the binding. In the presence of CaCl2, RFI values (Figure 10) increased with increases in EYPC content, whereas in the absence of CaCl2, RFI values decreased with increases in EYPC content. Taken together with the spectra in Figure 9, the RFI data (Figure 10) show that increasing EYPC content without calcium ions reduces the extent of interactions of BDT with mixed micelles. In contrast, increasing EYPC content with calcium ions markedly promotes micellar binding as opposed to aqueous self-association as evidenced by BDT fluorescence quenching. Because mixed micellar sizes are not altered by 10 and 15 mM CaCl2 in these experiments (Table 1), both BS and OH groups as well as phosphylcholine groups of PC are involved in mixed micellar− BDT binding. Effect of Calcium on Interactions of BDT with SUVs. From our UV−vis data with various neutral electrolytes, it became apparent that calcium ions play a specific if not unique role in the interactions of BDT with SUVs (Figure 11). What was observed was not a pure counterion effect because other divalent cations (Mg and Mn) added at concentrations 5 times the Ca2+ concentration yielded only subtle changes in the BDT/SUV spectra (Figure 11). It became clear from the UV− vis study that small increases in calcium dication concentration (Figure 12) as small as 1 mM promoted the binding of 50 μM

the isosbestic point, the spectrum of pure UCB is not included. Therefore, these results taken together suggest that the strong affinity of membrane lipids, principally long-chain PCs,43 for binding ionized UCB molecules is driven principally by interactions between membrane lipid headgroups and the anionic groups of UCB. In striking contrast, when UCB is essentially undissociated at pH 7.4,36,38,44 the nonionic species is dissolved hydrophobically within the bilayers of long-chain PCs.45 Interactions of BDT with BS (NaTC)/EYPC micelles (Figure 9a) were dominated by partitioning of BDT monomers onto a hydrophilic environment of the mixed micelles. Because of the similarity of the BDT mixed micellar spectra to that of BDT with pure NaTC (curve 2 in Figure 9a), BDT molecules appear to remain bound to the α-oriented OH functions on the hydrophilic surface of the BS, the same as that postulated earlier by us for simple BS micelles.1 Progressive increases in EYPC content exhibited two effects: enlarging the micelles after a certain critical ratio is reached46 and decreasing the non-PCassociated BS, which in this present case consists of monomers and simple micelles of NaTC.47,48 In studies39 of the NaTDC/ EYPC systems (3 g/dL, 0.15 M NaCl, 23 °C, pH 7.0) with 200 μM BDT, extending to equimolar NATDC:EYPC ratios, i.e., close to the mixed micellar phase limit,46 the hypsochromic λmax at 417 nm is more pronounced but no shift occurred with increasing EYPC:NaTDC ratios. Hence, with increasing EYPC:BS ratios in mixed micelles with NaTDC38,39 or with NaTC as in the work presented here (Figure 9a), there is a larger relative distribution of BDT onto polar domains of the mixed micelles as indicated by a higher ratio of bound BDT (A419) to unbound BDT (A450), principally as aqueous monomers. Effects of Calcium on Interactions of BS Micelles with BDT. As demonstrated in Figure 4, BDT exhibited an extremely high binding affinity for calcium, which on an equimolar basis was 2.5-fold greater than with taurine-conjugated BS with formation of soluble complexes.7 Whereas studies with pure BDT were conducted with 8 mM CaCl2 and 126 mM NaCl, we wished to observe the most exaggerated counterion effects on both BS and BDT by employing 50 mM CaCl2 in the spectrophotometric studies. It became clear (Figure 5) that 50 mM calcium in contrast to 150 mM NaCl favored the micellar partitioning of BDT irrespective of whether the CMC was critical (NaTDC) or uncritical (NaTC).9,35 As displayed in Figure 6, 50 mM CaCl2 was much more potent than 150 mM NaCl (see Figure 5 and ref 1) in quenching BDT fluourescence (Figure 6a,b). This most likely occurred because of a shift in the monomer−dimer−tetramer equilibrium (curve 1 in Figure 9b) to the more aggregated state as a result of screening the sulfonate charges of BDT by Ca2+ binding (Figure 4). Calcium binding by taurine-conjugated BS to form soluble complexes is well-documented,7,13,47 and as exemplified in Table 1, strong micellar growth is promoted in NaTDC but not in NaTC systems. Two factors, (i) the sharp increase in fluorescence quantum yield when NaTDC and NaTC achieved micellar concentrations of ∼1−2.5 and ∼2−5 mM, respectively, in 50 mM CaCl2 (Figures 5 and 6) and (ii) RFI values that showed no significant change with monomeric BS concentration (Figure 6), suggested distinct physicochemical effects. First, it implies an inhibition of BDT self-aggregation by BS micelles (Figure 9a), thereby preventing the strong self-quenching of BDT in the calcium environment and, second, as shown by the anisotropy ratios, increasing the microenvironmental mobility 6792

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cBR−Ca−SUV complexes. These may be of crucial importance for binding calcium and calcium-sensitive anions of bile pigment and, thus, preventing Ca-bilirubinate [as Ca(HUCB)2] as well as calcium carbonate precipitation.12,13 One of the principal pathways for the crystallization of cholesterol from bile occurs from cholesterol-rich vesicles (reviewed in ref 54 and 55). Aggregation and/or fusion of vesicles appears to be an important step in the cascade of cholesterol precipitation. Because the work presented here suggests that binding of cBR to unilamellar vesicles is Ca2+catalyzed and vesicles would provide a surface for βglucuronidase hydrolysis, this might provide a conceptual framework for understanding why the nucleus of cholesterol gallstones invariably contains high levels of Ca(HUCB)2 and inorganic calcium salts.12,56 Taken together with our earlier work,1,46 the observations presented here suggest that free Ca2+ and cBR are bound in solution by both simple and mixed micelles and SUVs and that Ca2+ and cBR induce aggregation but not fusion of vesicles. Because cholesterol molecules show a preference for the interior of polyunsaturated lipid membranes57 and are buried in the cores of mixed micelles modeling human bile,58 cholesterol is unlikely to influence any of our findings herein appreciably. The correctness of these concepts will need to be tested when gram quantities of bilirubin 1-O-glucuronosides (the principal human cBRs are the two constitutional isomers of BMG and BDG) become available for biophysical and chemical studies. In the past, BDG has been isolated from human gallbladder bile and biosynthesized with fresh liver homogenates for in vitro experiments.59 However, BDG is difficult to prepare and purify from other biliary components, and it is intrinsically very unstable because of isomeric scrambling of the OH-COOH functions from 1-O-acylglucuronosides into 2-, 3-, and 4-Oacylglucuronosides.60,61 The fact that of all the polyvalent cations in bile,39 only calcium may be specific for internalization of cBRs within the vesicular lipid bilayer of PC is an intriguing observation stemming from this work. A physicochemical disequilibrium between Ca2+ and cBRs in cholesterol-poor bile and cholesterol-rich vesicles in cholesterol supersaturated bile may initiate the cascade to either pigment or cholesterol gallstone formation. Moreover, it might be possible that the transport of cBR, particularly the highly polar BMG and BDG molecules, through membranes of the lower hepatobiliary and alimentary systems might occur with calcium as a neutralizing “trigger”. Nonetheless, the two sulfonate moieties in BDT and the two carboxylate moieties in BDG may respond very differently in their physicochemical behavior to the binding of Ca2+. Accordingly, we speculate that the vesicular aggregation preceding cholesterol and perhaps even Ca(HUCB)2 precipitation initiating the cascade leading to gallstone formation may require the presence of both calcium and at least partially ionized UCB and cBRs.

BDT and 1 mg/mL SUVs and that this was augmented progressively by increasing CaCl2 concentrations of up to 20 mM. In addition to the UV−vis studies, the QLS data in Table 2 indicate that whereas CaCl2 alone (to 30 mM) produced no influence on SUV size, calcium caused SUVs to grow by 55 Å with 50 μM BDT. Table 3 illustrates that the combination of both calcium and BDT was absolutely necessary to induce SUV growth; 250 μM BDT alone had no appreciable effect on SUV size (or spectral properties of the solutions), but the combination of 5 mM CaCl2 and 250 μM BDT increased SUV sizes from 115 to 270 Å. Progressive increases in RFI values of 40 μM BDT in proportion to added CaCl2 (Figure 13a) and the partial nullification of this effect with NaCl suggest that the specific calcium-promoted BDT−SUV interactions take precedence over the counterion effect [as depicted with NaCl (Figure 13b)]. Added NaCl tended to lower RFI values most likely by promoting BDT self-association. One possible model for this interaction might be the formation of metastable water-soluble Ca−BDT salts, which are less water-soluble than BDT−Na alone (Figure 4). In fact, the experiments illustrated in Figure 4 suggest considerable metastability of BDT−Ca systems in that microprecipitation of calcium−BDT salts occurred in proportion to calcium content after 24 h. Therefore, these complexes might be internalized into the PC bilayer near the hydrophilic headgroup through hydrophobic interactions.45 Another possibility is a salt bridge between zwitterions of PC with Ca2+ and BDT. The resulting complex could subsequently form aggregates possibly via a BDT−Ca− BDT bridge that is reversible, and this is evidenced in the SUV disaggregation experiments when calcium was chelated with Na4EDTA (Figure 14). Whereas an aggregation phenomenon without fusion was not induced by other divalent cations, such as Mg or Mn, our findings clearly suggest a specific role for calcium in the interaction of cBR with PC vesicles and to some extent with PC in BS/EYPC mixed micelles. It is noteworthy that in this connection calcium dications are characterized by low hydration numbers49 compared to those of alkali metals and other alkali earth metals as well as Mn2+, a representative cation of group 7 in the periodic table. Pathophysiological and Biological Implications. Insoluble Ca anion salt precipitation is an important event in gallstone disease, especially pigment stone formation.6,12,14 Supersaturation but not necessarily nucleation occurs when the solubility product of the specific Ca anion salt is exceeded either by high levels of the free (unbound) anion of UCB or the free (unbound) ionized Ca2+. Although there is consensus50 about the critical threshold of the free (unbound) UCB anion (Bf) in human plasma/serum of ∼70 nM, such data have never been determined for UCB anions in bile. BS are not only solubilizers of the sparingly soluble UCB in bile36−38,44 but also important buffers for free ionized Ca2+,13,47 principally via BS/ PC mixed micelles.7 However, whether their capacity is sufficient to bind excess Ca2+ ions in bile and prevent the ion product of Ca2+ and Ca2+-sensitive anions from exceeding their solubility products has not been resolved. X-ray crystallographic and NMR studies reveal that in BS crystals and solutions, Ca2+ is bound to the ionic BS side chains in a bidentate ion−ion bond.51−53 The tight binding of BDT to BS adds two more negatively charged groups to the micellar BS system, which as shown in this work facilitates electrostatic interactions with Ca2+. The findings in our study are highly suggestive of additional solubilizers of free Ca2+ ions in bile in the form of BS−Ca−cBR mixed micelles and Ca−cBR plus

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AUTHOR INFORMATION

Corresponding Author

*Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115. E-mail: [email protected]. Fax: (617) 730-5807. Phone: (617) 732-5822. Present Address †

M.W.N.: Krankenhaus Maria Stern, Am Anger 1, Remagen 53424, Germany. 6793

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(10) Cohen, D. E., Kaler, E. W., and Carey, M. C. (1993) Cholesterol carriers in human bile: Are “lamellae” involved? Hepatology 18, 1522− 1531. (11) Donovan, J. M., and Carey, M. C. (1990) Separation and quantitation of cholesterol “carriers” in bile. Hepatology 12, 94S−105S. (12) Cahalane, M. J., Neubrand, M. W., and Carey, M. C. (1988) Physical chemical pathogenesis of pigment gallstones. Semin. Liver Dis. 8, 317−328. (13) Moore, E. W., Celic, L., and Ostrow, J. D. (1982) Interactions between ionised calcium and sodium taurocholate: Bile salts are important buffers for prevention of calcium-containing gallstones. Gastroenterology 83, 1079−1089. (14) Ostrow, J. D. (1990) Unconjugated bilirubin and cholesterol gallstone formation. Hepatology 12, 219S−226S. (15) Cohen, D. E., and Carey, M. C. (1990) Rapid (1hr) high performance gel filtration chromatography resolves coexisting simple micelles, mixed micelles and vesicles in bile. J. Lipid Res. 31, 2103− 2112. (16) Tausk, R. J. M., Karmiggelt, J., Oudshoorn, C., and Overbeek, J.Th.G. (1974) Physical chemical studies of short-chain lecithin homologues. I. Influence of the chain length of the fatty acid ester and of electrolytes on the critical micellar concentrations. Biophys. Chem. 1, 175−183. (17) Tausk, R. J. M., van Esch, J., Karmiggelt, J., Voordouw, G., and Overbeek, J.Th.G. (1974) Physical Chemical studies of short-chain lecithin homologues: II. Micellar weights of dihexanoyl-and diheptanoyllecithin. Biophys. Chem. 1, 184−203. (18) Haberland, M. E., and Reynolds, J. A. (1975) Interaction of L-αpalmitoyl lysophosphatidylcholine with the A1 polypeptide of high density lipoprotein. J. Biol. Chem. 250, 6636−6639. (19) Huang, C.-H., Lapides, J. R., and Levin, I. W. (1982) Phasetransition behavior of saturated symmetric chain phospholipid bilayer dispersions determined by Raman spectroscopy: Correlation between spectral and thermodynamic parameters. J. Am. Chem. Soc. 104, 5926− 5930. (20) Van Echteld, C. J. A., de Kruijff, B., Mandersloot, J. G., and deGier, J. (1981) Effects of lysophosphatidylcholines on phosphatidylcholine and phosphatidylcholine/cholesterol liposome systems as revealed by 31P-NMR, electron microscopy and permeability studies. Biochim. Biophys. Acta, Biomembr. 649, 211−220. (21) Ramsammy, L. S., and Brockerhoff, H. (1982) Lysophosphatidylcholine − cholesterol complex. J. Biol. Chem. 257, 3570−3574. (22) McDonagh, A. F. P., Palma, L. A., Lauff, J. J., and Wu, T. W. (1984) Origin of mammalian biliprotein and rearrangement of bilirubin glucouronides in vivo in the rat. J. Clin. Invest. 74, 763−770. (23) Pope, J. L. (1967) Crystallization of sodium taurocholate. J. Lipid Res. 8, 146−147. (24) Carey, M. C., and Koretsky, A. (1979) Self-association of unconjugated bilirubin IXa in aqueous solution at pH 10 and physicalchemical interactions with bile salt monomers and micelles. Biochem. J. 179, 675−689. (25) Barenholz, Y., Gibbes, D., Litman, B. J., Goll, J., Thompson, T. E., and Carlson, F. D. (1977) A simple method for the preparation of homogenous phospholipid vesicles. Biochemistry 16, 2806−2810. (26) Mazer, N. A., Carey, M. C., Kwasnick, R. F., and Benedek, G. B. (1979) Quasielastic light scattering studies of aqueous biliary lipid systems: Size, shape and thermodynamics of bile salt micelles. Biochemistry 18, 3064−3075. (27) Cohen, D. E., Fisch, M. R., and Carey, M. C. (1990) Principles of laser light scattering spectroscopy: Application to the physicalchemical study of model and native biles. Hepatology 12, 113S−121S. (28) Cohen, D. E., Angelico, M., and Carey, M. C. (1990) 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. (29) Knudsen, A. P., Pedersen, A. O., and Brodersen, R. (1986) Spectroscopic properties of bilirubin-human serum albumin complexes: A stochiometric analysis. Arch. Biochem. Biophys. 244, 273−284.

Supported in part by National Institutes of Health Grants DK36588 and DK34854 (M.C.C.), National Science Foundation Grants BBS 86-15815 and DIR9002027 (T.M.L.), and Deutche Forschungsgemeinschaft Fellowship Ne345/1-1 (M.W.N.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Drs. Martin Reers and Judith Storch are thanked for advice on fluorescence spectroscopy. Monika R. Leonard provided able technical assistance with the calcium electrode studies, and we acknowledge with thanks Rebecca L. Ankener, Elaine O’Rourke, Tara Traylor, and Roopwinder Pruthi for their superb editing and word-processing of the manuscript.

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ABBREVIATIONS BDT, bilirubin ditaurate; BS, bile salt; cBR, conjugated bilirubin; PC, phosphatidylcholine; Ca2+, ionized calcium; UCB, unconjugated bilirubin; LPC, lyso(sn-1-palmitoyl)phosphatidylcholine; diC6- and diC7-PC, dihexanoyl- and dihepatoylphosphatidylcholine, respectively; EYPC, egg yolk phosphatidylcholine; UV−vis, UV−visible spectrophotometry; SUVs, small unilamellar vesicles; NaTC, sodium taurocholate; NaTDC, sodium taurodeoxycholate; R̅ h, mean hydrodynamic radius; QLS, quasi-elastic light scattering; RFI, relative fluorescence intensity; λmax, wavelength (nanometers) of maximal absorption; Amax, absorbance at λmax; BMG, bilirubin monoglucuronoside; BDG, bilirubin diglucuronoside.

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DOI: 10.1021/acs.biochem.5b00874 Biochemistry 2015, 54, 6783−6795