Solubility of Cholesterol in Quaternary Mixtures of Bile Salt, Lecithin

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Solubility of Cholesterol in Quaternary Mixtures of Bile Salt, Lecithin, Inorganic Salt, and Water at 310.2 K Jie Lu,*,† Danhui Wu,† Lianwei Chen,† Lijuan Zhang,‡ and Shimin Mao§ †

School of Chemical & Material Engineering, Jiangnan University, Wuxi 214122, People’s Republic of China School of Chemistry & Chemical Engineering, Shanghai University of Engineering Science, Shanghai 201620, People’s Republic of China § Department of Chemical Engineering & Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada ‡

ABSTRACT: The solubility of cholesterol (Ch) in quaternary mixtures of bile salt (BS), lecithin (L), inorganic salt (Ca2+), and water, which can be used as model biles was measured at 310.2 K in this work. The accurate concentrations of bile salt, lecithin, and cholesterol were determined by high performance liquid chromatography (HPLC). The effect of bile salt/lecithin molar ratio, bile salt concentration, total lipid concentration, and Ca2+ concentration on the solubility of cholesterol were investigated in detail. The results show that the cholesterol solubility monotonically increases with the increase of the concentration of bile salt or total lipid. The cholesterol solubility first increases with the bile salt/lecithin molar ratio when the bile salt/lecithin molar ratio is lower than 1 and then decreases when the bile salt/lecithin molar ratio further increases. In addition, the existence of Ca2+ in the model biles will significantly decrease the solubility of cholesterol.

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INTRODUCTION In recent years, cholesterol cholelithiasis has been emerging as one of the most common digestive diseases, of which the prevalence rate has considerably increased up to about 10% as a result of increased prevalence of overweight and a higher proportion of the elderly in the population.1,2 Gallstones generally can be categorized into three major types,3 cholesterol stones (in which the content of cholesterol is more than 70% by weight), mixed stones (in which there simultaneously exist a wide variety of cholesterol and bilirubinates), and pigment stones (in which the main components are various bilirubinates and the content of cholesterol is less than 10% by weight).4−8 Sethi and Johnson9 have summarized the pathogenesis of gallstones as follows: when cholesterol exceeds the amount that can be solubilized by the micelles and vesicles in bile, the formation of cholesterol stones may take place; when there is an excess of pigments and other components in bile due to bacterial infection or other factors, they shall precipitate and form pigment stones; when fine crystals of cholesterol or other debris appear in bile, bacteria may settle down and reproduce on their surface which shall result in mixed gallstones.10 In addition, some proteins such as α1-acid glycoprotein,11 α1antichymotrypsin,12 haptoglobin,13 apoprotein14 in bile have been found to act as promoters or inhibitors of gallstone formation. Bile can be regarded as a kind of complex biofluid mainly composed of water (about 80 to 96% in mass); however, cholesterol is generally insoluble in aqueous media. In their studies on the physical chemistry of cholesterol solubility in bile, Carey and Small15 have first put forward a triangular coordinate system in which three vertices indicate pure © 2014 American Chemical Society

cholesterol, lecithin, and bile salt, respectively. With the change of their concentrations, the bile will finally equilibrate into one phase (micelles), two phases (micelles and vesicles, micelles and crystals), or three phases (micelles, vesicles, and crystals), and cholesterol is packed in the micelles and vesicles. Supersaturation and even crystallization of cholesterol shall take place when the amount of cholesterol secreted into bile is higher than which micelles and vesicles can completely solubilize.16,17 To determine in vitro the solubility of cholesterol in bile, previous model system was usually the mixture of four components, for example, bile salt, lecithin, cholesterol, and water.18 In this study, we adopted a quinary system that contained bile salt, lecithin, cholesterol, water, and the inorganic salt (Ca2+) as a model bile because Ca2+ has been found to have an effect on the crystallization of cholesterol.19 HPLC was employed to determine the equilibrium concentrations of bile salt, lecithin, and cholesterol.

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EXPERIMENTAL SECTION Materials. Egg yolk lecithin (IUPAC name: 1,2-diacyl-snglycero-3-phosphocholine), bile salt that we choose sodium taurcholate (IUPAC name: 3α,7α,12α-trihydroxy-5β-cholanoyl taurine) as the salt of biles, and cholesterol (IUPAC name: 3βcholest-5-en-3-ol) were obtained from Sigma-Aldrich Co. (St. Louis, MO) and used without further purification. Calcium chloride of analytical grade was purchased from Xiya Reagent Received: May 1, 2014 Accepted: July 21, 2014 Published: July 25, 2014 2614

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Solubility Measurement. After a 480-h incubation, the incubated solutions were filtered through 0.22-μm Milllipore filters, and the obtained filtrates were then dissolved into the appropriate amounts of methanol for the HPLC analyses of bile salt, lecithin, and cholesterol. Each solubility data point reported here is the average of more than five measurements with a 2.0% relative standard deviation. HPLC analyses were performed using a ZORBAX-SB column (Agilent Technologies, Santa Clara, CA) with a flow rate of 1.0 mL·min−1 in isocratic elution mode. The wavelength of the G1314B detector was set at 210 nm. The column temperature was at 310.2 K. Mobile phases were prepared by methanol, acetonitrile, sodium acetate/phosphate buffer (pH 4.3) in a volumetric ratio of 73:20:7. Before each run, the HPLC column was first equilibrated with the mobile phase for 20 min. Then, the content of lecithin, bile salt, and cholesterol in different phases were analyzed after they were diluted by the mobile phases. The standard curves were obtained using pure chemicals. The instrument accuracy of the method was evaluated by performing five repetitive injections of pure chemicals’ standard solution (20 μL each time) and can be considered acceptable only when the peak area relative standard deviation was less than 2.0%. Statistical Analysis. Statistical analysis of the cholesterol solubility was performed by analysis of variance to determine whether difference existed among the solubility data at each specified time of examination. Statistical analysis of data was using one-factor analysis of variance.23

Co. (Shanghai, China). Deionized ultrapure water was prepared in-house with a Milli-Q water system (Millipore, Billerica, MA). Ultrafilters with a molecular weight cutoff (MWCO) of 10 000 and 300 000 were purchased from Millipore Co. (Billerica, MA), and dialysis membranes with an MWCO of 300 000 were from Spectrum Laboratories, Inc. (Rancho Dominguez, CA). The description of the materials used in this work is given in Table 1. Table 1. Suppliers and Mass Fraction Purity of the Materials component

suppliers

mass fraction

analysis method

bile salt lecithin cholesterol calcium chloride methanol chloroform

Sigma-Aldrich Co. Sigma-Aldrich Co. Sigma-Aldrich Co. Xiya Reagent Co. Sinopharm Group Co. Sinopharm Group Co.

0.990 0.900 0.990 >0.990 >0.995 >0.995

BRa ARb ARb ARb AR,b HPLCc AR,b HPLCc

a

Biological reagent. chromatography.

b

Analytical reagent. cHigh-performance liquid

Preparation of Model Biles. Model bile solutions were prepared according to the method as previously described in the literature.20−22 First, aliquots of cholesterol which is dissolved in methanol, lecithin in chloroform−methanol 2:1 (v/v), and bile salt in methanol were added to a volumetric flask at the desired lipid levels. Second, the organic solvents were evaporated under a stream of nitrogen at 313.2 K until the residual mixtures were condensed to viscous pastes, which were further freeze-dried to achieve a complete solvent removal. Then, the required volume of Tris (hydroxymethyl) aminomethane buffers (with the concentrations of Tris and NaCl of 0.025 and 0.150 mol·kg−1 respectively, with pH of 7.5) containing different dosages of calcium chloride was added to the freeze-dried mixtures. Next, the resulting solutions were flushed with dried nitrogen until microscopically isotropic and shaken at a constant speed of 100 rpm in a HYL-A shaker (Qiangle Laboratory Equipment Co., Taicang, China) at 328.2 K. Finally, the solutions were filtered through the MWCO and moved to a DRP 9082 incubator (Sumsung Laboratory Instrument Co., Shanghai, China) for being incubated at 310.2 K.

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RESULTS AND DISCUSSION There are many factors which can affect the solubility of cholesterol in biles, such as bile salt/lecithin molar ratio, Ca2+ concentration, total bile salt concentration, total lipid concentration, degree of cholesterol supersaturation, protein type, and concentration.19,24 In the present study, the effect of bile salt/lecithin molar ratio, bile salt concentration, total lipid concentration, and Ca2+ concentration in the model biles on the cholesterol solubility has been obtained, and the ternary phase diagram indicating phase equilibrium is finally drafted as well. Effect of Bile Salt/Lecithin Molar Ratio on Cholesterol Solubility. Generally, bile salts are synthesized in the liver from cholesterol and secreted into bile. In the course of this

Table 2. Cholesterol Solubility in Model Biles, Each with Different Bile Salt:Lecithin Ratios As a Function of the Total Lipid Concentration at Temperature T = 310.2 K, Pressure P = 0.1 MPaa,b composition of model biles mBS

c

mL

d

mChe

mTf

rg

× 10−3 mol·kg−1

× 10−3 mol·kg−1

× 10−3 mol·kg−1

× 10−3 mol·kg−1

117.4 117.4 176.2 88.1 117.4 176.2 117.4 187.9 234.9

45.1 28.6 0 88.1 38.8 0 117.4 45.6 0

18.0 12.6 19.5 19.5 15.2 19.5 26.0 20.4 26.0

81.0 81.0 81.0 162.1 162.1 162.1 405.2 405.2 405.2

sa × 10−3 mol·kg−1

2.6 4.1 1:0 1 3.0 1:0 1 4.1 1:0

4.545 4.021 2.125 9.399 7.291 3.401 23.564 16.986 8.698

a Solubility, s; P < 0.05, compared with control. bStandard uncertainties u are u(T) = 0.1 K, u(p) = 0.005 MPa, and u(s) = 0.0001 mol·kg−1. cmBS is the concentration of bile salt. dmL is the concentration of lecithin. emCh is the concentration of cholesterol. fmT is total lipids. gr is the molar ratio of bile salt to lecithin.

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Table 3. Cholesterol Solubility in Model Biles (Each with Fixed Total Lipid Concentration of 121.4 ×10−3 mol·kg−1) as a Function of Bile Salt/Lecithin Molar Ratio at Temperature T = 310.2 K, Pressure p = 0.1 MPaa,b composition of model biles mBSc

mLd

mChe

rf

× 10−3 mol·kg−1

× 10−3 mol·kg−1

× 10−3 mol·kg−1

62.9 88.1 116.4 117.4 117.4 117.4 146.8

125.9 88.1 65.6 45.1 38.8 28.6 17.8

21.0 19.5 15.7 17.8 15.4 12.6 6.95

sa × 10−3 mol·kg−1

0.5 1.0 1.8 2.6 3.0 4.1 8.2

6.866 7.324 7.847 7.357 5.068 7.428 3.139

Solubility, s; P < 0.05, compared with control. bStandard uncertainties u are u(T) = 0.1 K, u(p) = 0.005 MPa, and u(s) = 0.0001 mol·kg−1. cmBS is the concentration of bile salt. dmL is the concentration of lecithin. emCh is the concentration of cholesterol. fr is the molar ratio of bile salt to lecithin. a

Table 4. Cholesterol Solubility in Model Bilesg As a Function of Bile Salt/Lecithin Molar Ratio at Temperature T = 310.2 K, Pressure P = 0.1 MPaa,b composition of model biles mBSc × 10

−3

mol·kg

65.1 88.1 116.4 146.8 146.8 152.7 176.2

mLd −1

−3

× 10

mol·kg

mChe −1

−3

× 10

130.0 88.1 65.6 56.5 48.7 37.2 21.4

mol·kg

21.6 19.5 15.5 22.6 18.8 16.4 8.34

rf −1

sa × 10

0.5 1 1.77 2.6 3.02 4.1 8.2

−3

mol·kg−1

14.035 14.712 11.589 11.188 10.298 8.850 6.088

Solubility, s; P < 0.05, compared with control. bStandard uncertainties u are u(T) = 0.1 K, u(p) = 0.005 MPa, and u(s) = 0.0001 mol·kg−1. cmBS is the concentration of bile salt. dmL is the concentration of lecithin. emCh is the concentration of cholesterol. fr is the molar ratio of bile salt to lecithin. g Each with fixed total lipid concentration of 243.0 ×10−3 mol·kg−1. a

Figure 1. Effect of BS:L molar ratio (r) on cholesterol solubility (s) in model bile system with the total lipid concentration of 202.4 × 10−3 mol·kg−1.

Figure 2. Effect of total lipid concentration (mT) on cholesterol solubility (s) in model bile system with different BS:L molar ratios (r): ■, r = 1; ▲, r = 2.6; ⧫, r = 4.1; □, r = 8.2; Δ, r = 1:0.

synthesis, hydroxyl groups are added to the sterol ring and a carboxyl group to the side chain of the cholesterol molecule. Normally these hydroxyl and carboxyl groups will align along one side of the bile salt molecule.25 Therefore, a bile salt molecule can be regarded as a plate on which one side is hydrophilic and the other side is hydrophobic. Bile salt monomers are relatively soluble in water with a solubility of approximately 10−3 mol·kg−1, which is much higher than that of cholesterol or lecithin monomers. Above this concentration, bile salt monomers shall aggregate into simple micelles, in

whose center there is a steroid nucleus and in the outside there are hydrophilic groups. Therefore, cholesterol can be dissolved into bile with the help of simple micelles of bile salts. Lecithin, the major phospholipid of bile, has a backbone in which glycerol is linked to two fatty acid chains and a choline group. Thus, lecithin belongs to a linear amphipathic molecule that has a hydrophilic head and a hydrophobic body and tail. Moreover, lecithin molecules can be incorporated into simple 2616

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Figure 3. Effect of bile salt concentration (mBS) on cholesterol solubility (s) in model bile system (each with a fixed total lipid concentration of 162.1 × 10−3 mol·kg−1) with different BS:L molar ratios (r): ■, r = 1; ●, r = 3.0; ▲, r = 4.1; ▼, r = 8.2.

Figure 4. Effect of Ca2+ concentration on cholesterol solubility (s) in model bile system with the total lipid concentration of 303.5 × 10−3 mol·kg−1, every line means different Ca2+ concentration: ■, 0 mol· kg−1; ●, 2.5 × 10−3 mol·kg−1; ▲, 5 × 10−3 mol·kg−1.

bile salt micelles to form mixed micelles, and the latter might ripen into hollow spheres (called vesicles) at an appropriate ratio of bile salt to lecithin. In this case, cholesterol is readily incorporated into the lecithin bilayer of vesicles and there becomes associated with highly hydrophobic fatty acid chains. Table 2 has shown that cholesterol solubility in simple micelles is low, but mixed micelles and vesicles have a much greater

capacity to solubilize cholesterol, which can be attributed to the high affinity of cholesterol for the fatty acid chains of phospholipids.26 Tables 3 and 4 and Figure 1 show that the effect of bile salt/ lecithin molar ratio on cholesterol solubility in bile. As the increase of the molar ratio from zero to one, the solubility of cholesterol increases gradually; however, when the molar ratio

Table 5. Cholesterol Solubility in Model Biles with Different Ca2+ Concentrationsh As a Function of Bile Salt/Lecithin Molar Ratio at Temperature T = 310.2 K, Pressure P = 0.1 MPaa,b composition of model biles mLd

mChe

× 10−3 mol·kg−1

× 10−3 mol·kg−1

× 10−3 mol·kg−1

63.0

125.9

21.0

0.5

88.1

88.1

19.5

1.0

116.4

65.6

15.7

1.8

117.4

45.1

18.0

2.6

117.4

38.8

15.2

3.0

117.4

28.6

12.6

4.1

146.8

17.8

176.2

0

mBS

c

rf

7.08

8.2

19.5

1:0

mCag

sa

× 10−3 mol·kg−1

× 10−3 mol·kg−1

0 2.5 5.0 0 2.5 5.0 0 2.5 5.0 0 2.5 5.0 0 2.5 5.0 0 2.5 5.0 0 2.5 5.0 0 2.5 5.0

9.319 9.286 7.913 9.515 9.286 9.057 7.946 7.675 7.494 7.847 7.814 7.178 7.520 7.291 6.875 6.997 6.604 5.559 4.479 4.185 3.989 3.597 3.401 3.139

a Solubility, s; P < 0.05, compared with control. bStandard uncertainties u are u(T) = 0.1 K, u(p) = 0.005 MPa, and u(s) = 0.0001 mol·kg−1. cmBS is the concentration of bile salt. dmL is the concentration of lecithin. emCh is the concentration of cholesterol. fr is the molar ratio of bile salt to lecithin. g mCa is the concentration of calcium chloride. hEach with fixed total lipid concentration of 162.1 × 10−3 mol·kg−1.

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bile salt concentration increases progressively with the lecithin content up to a bile salt: lecithin molar ration of 1:1. This is because, with the increase of bile salt concentration, the number of micelles or mixed micelles increases. Effect of Ca2+ Concentration on Cholesterol Solubility in Bile. The addition of Ca2+ to model biles generally can affect the cholesterol solubility when the biles are sufficiently diluted so as to contain plentiful isolated small vesicles. As shown in Table 5 and Figure 4, different Ca2+ concentrations (2.5 × 10−3 mol·kg−1 and 5 × 10−3 mol·kg−1) have effects to different extents on the cholesterol solubility but present a certain trend. In general, when Ca2+ concentration is increased, the cholesterol solubility decreases. Bile salt has free radicals of R-COO−, meanwhile, the metal cation has the opposite charge so that a kind of complexation of the bile salt anion with the metal ion will take place that shall destroy the structure of micelles or vesicles in model biles, leading to the decrease in the solubilization of bile salt and the solubility of cholesterol in the dispersed system. At the same time, divalent cations, in particular Ca2+, have strong interaction with negatively charged lecithin vesicles, leading to increased permeability along with aggregation and fusion.27,28 Phase Diagram. Figure 5 shows solubility curves with different total lipid concentrations. The region under the curve is called the one phase zone which contains only micelles. When the total lipid concentration is increased, the area of one phase zone (micelles) shall be enlarged. Besides, as shown in Figure 6, with the increasing of Ca2+ concentration, the one phase zone (micelles) becomes smaller, which means that the solubility and crystal nucleation time of cholesterol will be reduced.29

Figure 5. Triangular phase diagram of the model biles consisting of bile salt, lecithin, cholesterol, water, Ca2+ (2.5 × 10−3 mol·kg−1) with different total lipid concentrations, every line means different total lipid concentration: ⧫, 81.0 × 10−3 mol·kg−1; ▲, 202.4 × 10−3 mol· kg−1; ■, 303.5 × 10−3 mol·kg−1; ●, 405.2 × 10−3 mol·kg−1.

Figure 6. Triangular phase diagram of the model biles consisting of bile salt, lecithin, cholesterol, water, Ca2+ with the total lipid concentration of 303.5 × 10 −3 mol·kg −1 and different Ca 2+ concentrations: ●, 0 mol·kg−1; ■, 2.5 × 10−3 mol·kg−1; ▲, 5 × 10−3 mol·kg−1.

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further increases, the solubility decreases apparently. As stated above, bile salt molecules can aggregate into simple micelles in which the lipophilic molecules can dissolve the cholesterol insoluble in water. When lecithin is added into a bile salt solution, they will form a kind of bile salt−lecithin mixed micelle. Generally 1 mol bile salt can dissolve 2 mol lecithin, that is, there are about 62 bile salt molecules and 125 lecithin molecules in a mixed micelle, which has more ability to dissolve cholesterol. When bile salt/lecithin molar ratio is larger than 1, as less lecithin and more bile salt exist in the bile, less cholesterol can be dissolved because less salt−lecithin mixed micelles will form in which lecithin-rich systems have a higher affinity of cholesterol than the simple micelles (i.e., aggregated bile salt molecules). When bile salt/lecithin molar ratio is less than 1, as less lecithin and more bile salt exist in the bile, more cholesterol can be dissolved because more simple micelles and/ or salt−lecithin mixed micelles will form. Effect of Total Lipid Concentration on Cholesterol Solubility in Bile. It was suggested earlier that the concentration of total lipids (bile salt + lecithin + cholesterol)15 might influence the maximum degree of cholesterol solubility in bile. In Figure 2, at all bile salt/lecithin molar ratios, cholesterol solubility markedly increases with the total lipid concentration in the range of (81.0 to 405.2) × 10−3 mol·kg−1. With the increase of total lipid concentration, the content of water is decreased meanwhile the contents of bile salt and lecithin are increased relatively, which will result in increased amount of micelles and vesicles. Effect of Total Bile Salt Concentration on Cholesterol Solubility in Bile. As shown in Figure 3, in the quinary system of model bile with the total lipid of 162.1 × 10−3 mol·kg−1, the increment in cholesterol solubility upon the increment in total

CONCLUSIONS HPLC has been employed in this work to determine the solubility of cholesterol in the quaternary mixtures of bile salt, lecithin, inorganic salt, and water at 310.2 K. The results clearly demonstrate that the solubility can be distinctly influenced by the composition of mixtures. Generally, a high concentration of bile salt and total lipids can enhance the dissolution ability of cholesterol. The solubility increases with the molar ratio of bile salt to lecithin when it is lower than 1, then decreases with its increase when higher than 1. Furthermore, the addition of Ca2+ will result in a lower solubility of cholesterol.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86 510 8591 7763. Notes

The authors declare no competing financial interest. The grants from the National Natural Science Foundation of China (Nos. 21176102 & 21176215), the Natural Science Foundation of Jiangsu Province (No. BK20131100), and the Sino-German Center for Research Promotion (No. GZ935) are sincerely acknowledged.

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