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The effect of modifications of lecithin and cholesterol on the micellar solubilization of cholesterol
407
BIOCHIMICA ET BIOPHYSICA ACTA BBA
56077
THE EFFECT OF MODIFICATIONS
OF LECITHIN AND CHOLESTEROL
THE MICELLAR SOLUBILIZATION
OF CHOLESTEROL*
DEWEY
P. ROTH
H. NEIDERHISER
AND
HAROLD
Medical Service, Veterans Administration chemistry, Case Western Reserve University (Received
February
Hospital, and the Department of Medicine School of Medicine, Cleveland, Ohio 44106
ON
and Bio(U.S.A.)
zest, 1972)
SUMMARY I. Differences in the degree of unsaturation of lecithin and lysolecithin had little effect on the quantity of cholesterol solubilized by micellar solutions of bile salts and lecithin or lysolecithin. A lecithin containing 83% unsaturated fatty acids was isolated from cabbage. This lecithin, when added to solutions containing 180 mM sodium taurocholate, solubilized 0.35 f 0.02 mmole of cholesterol per mmole of lecithin; an amount similar to that solubilized by egg lecithin (0.37 f 0.02 mmole) and human bile lecithin (0.39 f 0.04 mmole) which contain only 50% unsaturated fatty acids. 2. Cabbage lysolecithin contained 61% unsaturated fatty acids. This lysolecithin, when added to solutions containing 180 mM sodium taurocholate, solubilized as muchcholesterol (0.20 f 0.01 mmole) as the lysolecithin from egg (0.19 f 0.01 mmole) which contains only saturated fatty acids. 3. Solutions of taurocholate and lecithin solubilized less cholestanol (0.29 & 0.01 mmole) than cholesterol (0.37 f 0.02 mmole) and considerably less cholesteryl acetate (0.04 & 0.01 mmole).
INTRODUCTION
Cholesterol, the principal component of most human gallstones, is solubilized in bile by the micellar combination of lecithin and bile salts2*s.Lecithin itself is insoluble in water but is solubilized in bile by bile saltsa. Cholesterol in turn is solubilized by mixed micelles of lecithin and bile salts ag3.A change in the type of bile salt does not markedly affect the quantity of cholesterol solubilized by added lecithin’. It is uncertain whether a change in the type of lecithin has an effect. In an early study, Blomstrand and Ekdah16 suggested that the type of lecithin might be important; bile of patients with gallstone disease contained less poly-unsaturated fatty acids than bile of normal patients. Recently, Saunders and Wells6 using model bile solutions also * A preliminary report of this work has been published in abstract forml. BiocMm.
Biophys.
Acta,
270 (1972) 407-413
D. H. NEIDERHISER, H. I’. ROTH
408
found some differences in the cholesterol solubilizing powers of different synthetic and natural lecithins. However, in the report of Blomstrand and Ekdah15 and Saunders and Wells6 the number of observations were too small to establish whether these differences were significant. In this study, cabbage lecithin was compared with human bile and 83% as thin, in lecithin
egg lecithin. The proportion of unsaturated fatty acid in cabbage lecithin is compared to 500// in egg and human bile lecithins. Cabbage provided a leciample amounts, for study of the effect of the saturation of fatty acids in on cholesterol solubilization.
Lysolecithin is occasionally found in human gallbladder bile’. The fatty acid of human bile lysolecithin is normally saturateda. In this study we prepared a lysolecithin from cabbage lecithin which contained 61 y. unsaturated fatty acids and compared its ability to solubilize cholesterol, when added to a bile salt solution, with that of a lysolecithin that contained only saturated fatty acids. Nakayamao found cholesterol esters in human gallstones. M’e therefore studied the solubilization of cholesterol esters by micellar solutions of lecithin and bile salts. This presented some evidence for the relation of the hydroxyl group to cholesterol solubilization. In addition, in an effort to explore the effect of minor changes in the sterol structure on cholesterol solubilization, the solubility of cholestanol, a reduction product
of cholesterol,
was determined.
MATERIALS
Lecithins. Chromatographically pure egg lecithin was isolated as previously describedlo. Chromatographically pure cabbage leaf lecithin was isolated according to the method of Wheeldonlr. Gallbladder bile lecithin was isolated from samples of normal human gallbladder bile obtained at surgery. Four separate samples of gallbladder bile were extracted with chloroform-methanol (4: I, v/v) and the lecithin was then separated from the other lipids by chromatography on a silicic acid column with a chloroform:methanol gradient as previously described for egg lecithinlO. Lysolecithins. Egg and cabbage lysolecithin were prepared from egg and cabbage lecithins respectively according to the method of Hanahan I2 with snake venom (Naja naja) as the source of phospholipase A. The lysolecithin fatty acid mixtures were separated on silicic acid columns with a chloroform-methanol gradientlo. Sterols. Cholesterol (Nutritional Biochemicals, Cleveland, Ohio) was recrystallized 3 times from 95 o/oethanol and dried in a vacuum oven. The resulting cholesterol, sterols by thin-layer n1.p. 149-150 “C (lit.13 149 “C), was free from contaminating chromatography (Silica Gel G plates developed with cyclohexane-ethyl acetate-water, 60 : 40 :I, by vol.)l4. Cholestanol (Nutritional Biochemicals) was recrystallized 3 times from 95% ethanol and dried in a vacuum oven. The resulting cholestanol, m.p. 142-143 "c, (lit.l3142 “C) was found to be free of contaminating sterols by thin-layer chromatography14. Cholesteryl acetate was prepared by acylation of cholesterol in pyridine with acetic anhydride 15. Cholesteryl acetate, m.p. 115-116 “C (lit.13 116 “C), was found to be free of digitonin precipitable cholesterol. Cholesteryl oleate (Applied Science Labs., State College, Pa.) was found to be free of digitonin precipitable cholesterol. After alkaline hydrolysis, I mole of both cholesteryl acetate and cholesteryl oleate (by weight) gave I mole of cholesterol. Bile salt. Sodium taurocholate (Calbiochem, Lot 840219) was used as the test Uzochim.
Biophys.
Actn,
270 (1972) 407-413
SOLUBILIZATION
409
OF CHOLESTEROL
bile salt in all experiments. Thin-layer chromatographyls demonstrated that this lot contained less than 1% free bile acid. After alkaline hydrolysis”, I mole of sodium taurocholate gave I mole of cholic acid. METHODS
Analytical procedures. Phospholipids were separated by thin-layer chromatography and phospholipid phosphorus and phospholipid fatty acid were assayed as previously describeda. Cholesterol, after precipitation with digitonin, was assayed with the Liebermann-Burchard reagent as previously described*. Solutions containing cholesteryl esters were hydrolyzed in ethanolic KOH for 30 min at 65 “C16. After neutralization to a phenolphthalein end point with 10% acetic acid, the cholesterol was precipitated with digitonin and assayed as above. Cholestanol was assayed by the anthrone method of Vahouny et a1.18 after precipitation with digitonin. For the experiment on the solubilization of cholestanol, the amount of cholesterol solubilized in comparable experiments was determined with both the anthrone reagent and the Liebermann-Burchard reagent, Identical results were obtained with both of these analytical procedures. Measurement of sterol solubilization. To prepare a given model solution, sodium taurocholate (180 mM) was dissolved in 0.05 M phosphate buffer (pH 7.0). An ethanolic solution of the phospholipid was added to a IO ml erlenmeyer flask and the ethanol was evaporated in vacua at 40 “C. I ml of the taurocholate solution was then added and the lecithin residue dissolved. Excess crystalline cholesterol, cholestanol, or cholesterol ester (20 mg) sieved to give crystals ranging in size of from 0.45 to 44 pm (ref. IS), were added to a 2-ml glass ampoule. The taurocholate solution containing lecithin was added by micropipette to the ampoule, the mixture brought to a temperature of 37 “C, flushed with nitrogen, and then sealed in an atmosphere of nitrogen. The ampoules were then incubated at 37 “C with shaking for 4 days and without shaking for 2 day+. After incubation, the ampoule contents were filtered through a 0.22 ,um Millipore filter lo. Analytical determinations were made on the optically clear solutions obtained after Millipore filtration as previously describedIe. RESULTS
The fatty acid composition of lecithins and lysolecithins. The human bile lecithin isolated in these studies contained less stearic acid (C,,,,) and proportionately more palmitic acid (C,,,,) than the egg lecithin (Table I). Human bile lecithin also contained more polyunsaturated fatty acids. There was more linoleic (C,,,,) and arachidonic (C,,,,) acid and less oleic acid (C,,,,) than in the egg lecithin. Cabbage leaf lecithin contained more unsaturated fatty acids than either egg or bile lecithins. 83% of the fatty acids in cabbage lecithin were unsaturated (Table I), and 49”/0 of the total fatty acids were linolenic acid (C,,,,). While all of the fatty acids of egg lysolecithin were saturated (Table I), only 39% of the fatty acids in cabbage leaf lysolecithin were saturated. 50% of the total fatty acids of cabbage lysolecithin were polyunsaturated (Table I). Comparison of the cholesterol solubilizing ability of phospholi+Ls. Sixteen different concentrations of each lecithin (cabbage, human bile and egg), and each lysoleciBiochim.
Biophys.
Acta,
270
(1972)
407-413
410
D. H. NEIDERHISER,
TABLE
H. P. ROTH
I
FATTY ACID COMPOSITIONOF PHOSPHOLIPIDS Figures expressed as percentage
of total fatty acids.
Lecithins
Lysolecithins
Fatty acid
Gs:,
Egg*
Cabbage*
Human gallbladder bile* * K.C.
F.M.
W.B.
M.F.
32.9
15.8
36.5
42.4
1.8
1.8
45.8
41.8
3.3
3.5
4.5
12.8
1.6
31.8
9.3
5.4 16.6
5.2 14.8
4.9 3.8 15.2
3.9 17.1
22.1
26.4
30.0
20.7
26.4
11.8
4.1
9.6
6.3
16.3
cm,
49.4
C 20.4
Egg*
Cabbage *
73.3
33.4
26.7
6.0 11.4 25.0 24.2
4.4
* Values obtained from pooled preparations of egg and cabbage lecithins and lysolecithins. ** Values obtained from preparations of bile lecithins from 4 different patients (K.C., F.M., W.B., and M.F.).
thin (cabbage and egg) were added to 180mM sodium taurocholate solution and tested for their ability to solubilize cholesterol. Over a range of IO to 60 mmoles per 1, the range of lecithin observed in human gallbladder bile, cholesterol solubilization was proportional to the concentration of added phospholipid (Fig. I). The regression coefficient & standard deviation (SD.) of the regression coefficient of the line4 (values in parentheses) representing mmoles of cholesterol solubilized per mmole of added phospholipid was calculated by the method of least squares. Approximately the same quan-
3
HUMAN BILE LECITHIN
EGG LECITHIN (0.37i.02)
ITHIN (0.20f.01)
0
10
20
30
40
50
60
PHOSPHOLIPID (mmoles/liter)
Fig. I. Cholesterol solubilization by different concentrations of egg (A-A), cabbage (O-O) and bile (O-O) lecithins and egg (0-n) and cabbage (m-m) lysolecithins added to 180 mM sodium taurocholate. Crystalline cholesterol was incubated with the solutions for 6 days at 37 “C.Insoluble material was then removed with a 0.22 pm Millipore filter. The figures in parentheses represent the regression coefficients + S.D. of the regression coefficient of the lines and were calculated by the method of least squares. B&him.
Biophys.
Acta, 270 (1972) 407-413
SOLUBILIZATION
4x=
OF CHOLESTEROL
tity of cholesterol was solubilized by each of the lecithins when added to a 180 mM sodium taurocholate solution (Fig.) even though the egg, human bile and cabbage leaf lecithins differed markedly in their fatty acid composition. For every mmole of egg licithin added to a bile salt solution, 0.37 & 0.02 mmole of cholesterol was solubilized; for human bile lecithin 0.39 f 0.04 and for cabbage lecithin 0.35 rir:0.02 mmole Added cabbage and egg lysolecithins solubilized less cholesterol than their respective lecithins (Fig. I) but both lysolecithins solubilized approximately the same quantity of cholesterol. Cabbage lysolecethin, which contained 61 % unsaturated fatty accids, solubilized 0.20 & 0.01 mmole of cholesterol and egg lysolecithin which contained only saturated fatty acids solubilized 0.19 -& 0.01 mmole of cholesterol per mmole of added lysolecithin (Fig. I). E$ect of modification of cholesterol on its micellar solubilizatiolz. Cholestanol is a hydrogenated product of cholesterol and differs in that it does not have a double bond at CrCB. To test the effect this change in the structure of cholesterol has on its solub~zation, we measured the solub~zation of cholestanol by increasing concentrations of egg lecithin in 180 m&I solutions of sodium taurocholate. These solutions solubilized significantly smaller amounts of cholestanol than cholesterol (Fig. 2). One 30
CHOLESTERYL
5
0
10
ACETATE
20
30
LECITHIN
(mmol~r/lihc)
40
(0.04+.01)
50
60
Fig. 2. Cholesterol(o-o), cholestanol(&-A) and cholesterylacetate (O-O), solubilizationby differentconcentrationsof egg lecithin added to 180 mM sodiumtaurocholate.Conditionsof the experimentare the sameas in Fig. I. mmole of added lecithin solubilized 0.37 rfi.0.02 mmole of cholesterol but only 0.29 f 0.01 mmole of cholestanol. We also studied the solubility of cholesterol esters. The 180 mM solution of sodium taurocholate itself solubilized 1.2 mmole per 1 of cholesteryl acetate (Fig. 2). For every added mmole of lecithin, only 0.04 f o.or mmole of cholesteryl acetate was solubilized. Less than 0.1 mmole per 1 of cholesteryl oleate was solubilized by the bile %~%&a. Biophys. Acta,
270
(1972)
407-4~3
D. H.
412
NEIDERHISER,
H. P. ROTH
salt solution itself. No additional cholesteryl oleate was solubilized when lecithin was added to the bile salt solution. The solubilization of cholesterol, cholestanol and cholesteryl acetate was also tested by the method of Small d at .ao. r-ml aliquots of ethanolic solutions of 180 mM sodium taurocholate, 40 mM lecithin and 18 mM cholesterol or cholestanol or cholesteryl acetate were mixed, dried and the residue reconstituted with I ml of water. The solutions were allowed to equilibrate 20. After equilibration all of the cholesterol was in solution, however, only 14 mmole per 1 of cholestanol and 4 mmole per 1 of cholesteryl acetate was in solution; similar to the values reported above with our technique&. DISCUSSION
This study demonstrated that differences in the degree of unsaturation of lecithin had little effect on the quantity of cholesterol solubilized by a micellar solution of bile salts and lecithin. This suggests that differences in the degree of saturation of lecithin secreted in bile is unlikely to be important in the cause of gallstones in man. In the report of Blomstrand and Ekdahl& the fatty acid composition of lecithins from most patients with gallstones was within the range of fatty acid compositions recently reported in normal bilezf. The onlv abnormal samples were from z patients with occlusion of the cystic duct. In these biles, there tended to be less unsaturated fatty acids in the lecithins, but changes in the bile secondary to the occlusion itself could not be ruled out. The cabbage lecithin used in this study contained 830/ounsaturated fatty acids and 49% of the total fatty acids were linolenic acid (C,,,,) (Table I). When compared with egg and human bile lecithins which only contain 5o:/, unsaturated fatty acids and no linolenic acid (Table I), no difference in the cholesterol solubilized by the added lecithins was found (Fig. I). Hegardt and Dams2 produced an egg lecithin with increased unsaturated fatty acids by feeding chickens safflower oil. They also found no effect of the degree of unsaturation of the fatty acids of lecithin on cholesterol solubilization. Varying quantities of aracl~idollic acid (C,,,,) are found in human bile lecithin. However, neither our studies nor those of Hegardt and Dam2z or Saunders and Well9 have tested the possible solubilizing powers of lecithins containing major quantities of arachidonic acid. Since the quantity of this fatty acid in human bile is normally small (approximately 5 76 of the total fatty acids, Table I), it may not be important. When added to a bile salt solution, lysolecithin with one fatty acid solubilized only half as much cholesterol as did lecithin with z fatty acids (Fig. I). This suggested that the fatty acid was critical for the solubilization of cholesterol by these phospholipids. We anticipated that if the nature of the fatty acid of ~hos~Ilolipid was important, it should be most evident in our studies with Iysolecithin. Egg lysolecithin, like human bile lysolecithin, has no unsaturated fatty acids. It was possible to compare this saturated lysolecithin with the lysolecithin of cabbage which has 61~& unsaturated fatty acids. No difference in the cholesterol solubilizing powers of these lysolecithins, when added to a bile salt solution, was observed. This finding supports the concept that moderate changes in the saturation of fatty acids do not alter cholesterol solubilization by phospholipids. While changes in the fatty acids of the ~l~ospholi~id had no effect on cholesterol Biockim. Biopkys. z‘fcta, 270 (1972)
407-413
413
SOLUBILIZATION OF CHOLESTEROL
solubilization, motications of cholesterol did effect solubilization. Hydrogenation of the double bond of cholesterol decreased the so~ub~~at~on slightly but s~~i~~ant~~. There are several possible explanations, among them is a possible effect on the reactivity of the hydroxyl group. The possible significance of the hydroxyl group is also suggested by the diminished solubilization when esters are formed. Cholesteryl acetate was much less soluble than was cholesterol. Cholesteryl oleate was essentially insduble but here a long chain fatty acid had been added and marked changes would be expected. Significant quantities of cholesterol esters have not been demonstrated in bile. However, Nakayama@ has found a small amount of cholesterol esters in gallstones. The insolubility of the esters of cholesterol in mode1 bile solutions as observed in this study fits with their absence from bile and their presence in gallstones. ACKNOWLEDGEMENT
The authors thank Dr Ralph De Palma who provided the samples of human gallbladder bile. REFERENCES 1 2 3 4 5 6 7 8 g IQ IL IZ
13 r4 15 16
17 18 19 20 21 22
D. H. Neiderhiser and H. P. Roth, J. Lab. Clin. Med., 74 (1969) 493. A. F. Hofmann, G&roentemZogy, 48 (1965) 484” W. H. Admirand and D. M. Small, J. Clin. .i%vest., 47 (1968) 1043. D. H. Neiderhiser and H. P. Roth, Prac, Sac. &q%l. Bicrl. Med., 128 (1968) 221. R. Blomstrand and P. Ekdahl, Proc. Sot. Exptt. BioE. Med., x04 (1960) 205. D. R. Saunders and M. A. Wells, Be’oclrd%.Biopkys. A&a, 176 { fg6g) 828. A. Gottfries, S. Nilsson, B. Samuelsson and T. Schersttk, Scud. J. C&k La&. fwesd., z1{1g68j 168. 3. Borgstr&m, Acta C&em. Sand., II (~957) 749. F. Nakayama, J. Lab. Ctin. Med., 73 (1969) 623. D. H. Neiderhiser, H. P. Roth and L. T. Webster, J. Lab. CZilp. Med., 68 (1966) go. L. W. Wheeldon, J. Lipid Res., I (x960) 439, D. J. Hanahan, J. Biol. Ckem., 195 (rgp) 199. L. F. Fieser and M. Fieser, Steroids, Reinhold, New York, 1959, p. 28. R. D. Bennett and E. Heftmann, J. Ckromatog., 9 (1962) 359. A. H. Blat& Urga& Synbhssis, Wiley, New York, x943, p. 193. Il. Kritchevsky, D. S. Martak and G. H, Rothblat, Aarsl. B&&em., 5 (~963) 388. W. W. Wells and C, A. Baumann, A+x%. Biochem. Bi5#qs., 53 (1954) 471. G. V. Vahouny, C, R. Borta, R. M. Mayer and C. R. Treadwell, A%&. B&&e%, I (1960) 371. D. H. Neiderdiser and E-I.P. Roth, GasCroen&xaEogy,58 (1970) 26. D. M. Small, M, C. BourgBs and D. G. Dervichian, Biochim. Bio@ys. Acta, 125 (1966) 563. J. A. Balint, E. C. Kyriakides, H. L. Spitzer and E. S. Morrison, J. Lipid Res., 6 (1965) 96. F. G. Hegardt and H. Dam, Emahrungswissen, xo (x971) 223. Biochim. Bioj5bys.
Acta,
270 (1972) 407-4~3
BIOCHIMICA ET BIOPHYSICA ACTA BBA
56077
THE EFFECT OF MODIFICATIONS
OF LECITHIN AND CHOLESTEROL
THE MICELLAR SOLUBILIZATION
OF CHOLESTEROL*
DEWEY
P. ROTH
H. NEIDERHISER
AND
HAROLD
Medical Service, Veterans Administration chemistry, Case Western Reserve University (Received
February
Hospital, and the Department of Medicine School of Medicine, Cleveland, Ohio 44106
ON
and Bio(U.S.A.)
zest, 1972)
SUMMARY I. Differences in the degree of unsaturation of lecithin and lysolecithin had little effect on the quantity of cholesterol solubilized by micellar solutions of bile salts and lecithin or lysolecithin. A lecithin containing 83% unsaturated fatty acids was isolated from cabbage. This lecithin, when added to solutions containing 180 mM sodium taurocholate, solubilized 0.35 f 0.02 mmole of cholesterol per mmole of lecithin; an amount similar to that solubilized by egg lecithin (0.37 f 0.02 mmole) and human bile lecithin (0.39 f 0.04 mmole) which contain only 50% unsaturated fatty acids. 2. Cabbage lysolecithin contained 61% unsaturated fatty acids. This lysolecithin, when added to solutions containing 180 mM sodium taurocholate, solubilized as muchcholesterol (0.20 f 0.01 mmole) as the lysolecithin from egg (0.19 f 0.01 mmole) which contains only saturated fatty acids. 3. Solutions of taurocholate and lecithin solubilized less cholestanol (0.29 & 0.01 mmole) than cholesterol (0.37 f 0.02 mmole) and considerably less cholesteryl acetate (0.04 & 0.01 mmole).
INTRODUCTION
Cholesterol, the principal component of most human gallstones, is solubilized in bile by the micellar combination of lecithin and bile salts2*s.Lecithin itself is insoluble in water but is solubilized in bile by bile saltsa. Cholesterol in turn is solubilized by mixed micelles of lecithin and bile salts ag3.A change in the type of bile salt does not markedly affect the quantity of cholesterol solubilized by added lecithin’. It is uncertain whether a change in the type of lecithin has an effect. In an early study, Blomstrand and Ekdah16 suggested that the type of lecithin might be important; bile of patients with gallstone disease contained less poly-unsaturated fatty acids than bile of normal patients. Recently, Saunders and Wells6 using model bile solutions also * A preliminary report of this work has been published in abstract forml. BiocMm.
Biophys.
Acta,
270 (1972) 407-413
D. H. NEIDERHISER, H. I’. ROTH
408
found some differences in the cholesterol solubilizing powers of different synthetic and natural lecithins. However, in the report of Blomstrand and Ekdah15 and Saunders and Wells6 the number of observations were too small to establish whether these differences were significant. In this study, cabbage lecithin was compared with human bile and 83% as thin, in lecithin
egg lecithin. The proportion of unsaturated fatty acid in cabbage lecithin is compared to 500// in egg and human bile lecithins. Cabbage provided a leciample amounts, for study of the effect of the saturation of fatty acids in on cholesterol solubilization.
Lysolecithin is occasionally found in human gallbladder bile’. The fatty acid of human bile lysolecithin is normally saturateda. In this study we prepared a lysolecithin from cabbage lecithin which contained 61 y. unsaturated fatty acids and compared its ability to solubilize cholesterol, when added to a bile salt solution, with that of a lysolecithin that contained only saturated fatty acids. Nakayamao found cholesterol esters in human gallstones. M’e therefore studied the solubilization of cholesterol esters by micellar solutions of lecithin and bile salts. This presented some evidence for the relation of the hydroxyl group to cholesterol solubilization. In addition, in an effort to explore the effect of minor changes in the sterol structure on cholesterol solubilization, the solubility of cholestanol, a reduction product
of cholesterol,
was determined.
MATERIALS
Lecithins. Chromatographically pure egg lecithin was isolated as previously describedlo. Chromatographically pure cabbage leaf lecithin was isolated according to the method of Wheeldonlr. Gallbladder bile lecithin was isolated from samples of normal human gallbladder bile obtained at surgery. Four separate samples of gallbladder bile were extracted with chloroform-methanol (4: I, v/v) and the lecithin was then separated from the other lipids by chromatography on a silicic acid column with a chloroform:methanol gradient as previously described for egg lecithinlO. Lysolecithins. Egg and cabbage lysolecithin were prepared from egg and cabbage lecithins respectively according to the method of Hanahan I2 with snake venom (Naja naja) as the source of phospholipase A. The lysolecithin fatty acid mixtures were separated on silicic acid columns with a chloroform-methanol gradientlo. Sterols. Cholesterol (Nutritional Biochemicals, Cleveland, Ohio) was recrystallized 3 times from 95 o/oethanol and dried in a vacuum oven. The resulting cholesterol, sterols by thin-layer n1.p. 149-150 “C (lit.13 149 “C), was free from contaminating chromatography (Silica Gel G plates developed with cyclohexane-ethyl acetate-water, 60 : 40 :I, by vol.)l4. Cholestanol (Nutritional Biochemicals) was recrystallized 3 times from 95% ethanol and dried in a vacuum oven. The resulting cholestanol, m.p. 142-143 "c, (lit.l3142 “C) was found to be free of contaminating sterols by thin-layer chromatography14. Cholesteryl acetate was prepared by acylation of cholesterol in pyridine with acetic anhydride 15. Cholesteryl acetate, m.p. 115-116 “C (lit.13 116 “C), was found to be free of digitonin precipitable cholesterol. Cholesteryl oleate (Applied Science Labs., State College, Pa.) was found to be free of digitonin precipitable cholesterol. After alkaline hydrolysis, I mole of both cholesteryl acetate and cholesteryl oleate (by weight) gave I mole of cholesterol. Bile salt. Sodium taurocholate (Calbiochem, Lot 840219) was used as the test Uzochim.
Biophys.
Actn,
270 (1972) 407-413
SOLUBILIZATION
409
OF CHOLESTEROL
bile salt in all experiments. Thin-layer chromatographyls demonstrated that this lot contained less than 1% free bile acid. After alkaline hydrolysis”, I mole of sodium taurocholate gave I mole of cholic acid. METHODS
Analytical procedures. Phospholipids were separated by thin-layer chromatography and phospholipid phosphorus and phospholipid fatty acid were assayed as previously describeda. Cholesterol, after precipitation with digitonin, was assayed with the Liebermann-Burchard reagent as previously described*. Solutions containing cholesteryl esters were hydrolyzed in ethanolic KOH for 30 min at 65 “C16. After neutralization to a phenolphthalein end point with 10% acetic acid, the cholesterol was precipitated with digitonin and assayed as above. Cholestanol was assayed by the anthrone method of Vahouny et a1.18 after precipitation with digitonin. For the experiment on the solubilization of cholestanol, the amount of cholesterol solubilized in comparable experiments was determined with both the anthrone reagent and the Liebermann-Burchard reagent, Identical results were obtained with both of these analytical procedures. Measurement of sterol solubilization. To prepare a given model solution, sodium taurocholate (180 mM) was dissolved in 0.05 M phosphate buffer (pH 7.0). An ethanolic solution of the phospholipid was added to a IO ml erlenmeyer flask and the ethanol was evaporated in vacua at 40 “C. I ml of the taurocholate solution was then added and the lecithin residue dissolved. Excess crystalline cholesterol, cholestanol, or cholesterol ester (20 mg) sieved to give crystals ranging in size of from 0.45 to 44 pm (ref. IS), were added to a 2-ml glass ampoule. The taurocholate solution containing lecithin was added by micropipette to the ampoule, the mixture brought to a temperature of 37 “C, flushed with nitrogen, and then sealed in an atmosphere of nitrogen. The ampoules were then incubated at 37 “C with shaking for 4 days and without shaking for 2 day+. After incubation, the ampoule contents were filtered through a 0.22 ,um Millipore filter lo. Analytical determinations were made on the optically clear solutions obtained after Millipore filtration as previously describedIe. RESULTS
The fatty acid composition of lecithins and lysolecithins. The human bile lecithin isolated in these studies contained less stearic acid (C,,,,) and proportionately more palmitic acid (C,,,,) than the egg lecithin (Table I). Human bile lecithin also contained more polyunsaturated fatty acids. There was more linoleic (C,,,,) and arachidonic (C,,,,) acid and less oleic acid (C,,,,) than in the egg lecithin. Cabbage leaf lecithin contained more unsaturated fatty acids than either egg or bile lecithins. 83% of the fatty acids in cabbage lecithin were unsaturated (Table I), and 49”/0 of the total fatty acids were linolenic acid (C,,,,). While all of the fatty acids of egg lysolecithin were saturated (Table I), only 39% of the fatty acids in cabbage leaf lysolecithin were saturated. 50% of the total fatty acids of cabbage lysolecithin were polyunsaturated (Table I). Comparison of the cholesterol solubilizing ability of phospholi+Ls. Sixteen different concentrations of each lecithin (cabbage, human bile and egg), and each lysoleciBiochim.
Biophys.
Acta,
270
(1972)
407-413
410
D. H. NEIDERHISER,
TABLE
H. P. ROTH
I
FATTY ACID COMPOSITIONOF PHOSPHOLIPIDS Figures expressed as percentage
of total fatty acids.
Lecithins
Lysolecithins
Fatty acid
Gs:,
Egg*
Cabbage*
Human gallbladder bile* * K.C.
F.M.
W.B.
M.F.
32.9
15.8
36.5
42.4
1.8
1.8
45.8
41.8
3.3
3.5
4.5
12.8
1.6
31.8
9.3
5.4 16.6
5.2 14.8
4.9 3.8 15.2
3.9 17.1
22.1
26.4
30.0
20.7
26.4
11.8
4.1
9.6
6.3
16.3
cm,
49.4
C 20.4
Egg*
Cabbage *
73.3
33.4
26.7
6.0 11.4 25.0 24.2
4.4
* Values obtained from pooled preparations of egg and cabbage lecithins and lysolecithins. ** Values obtained from preparations of bile lecithins from 4 different patients (K.C., F.M., W.B., and M.F.).
thin (cabbage and egg) were added to 180mM sodium taurocholate solution and tested for their ability to solubilize cholesterol. Over a range of IO to 60 mmoles per 1, the range of lecithin observed in human gallbladder bile, cholesterol solubilization was proportional to the concentration of added phospholipid (Fig. I). The regression coefficient & standard deviation (SD.) of the regression coefficient of the line4 (values in parentheses) representing mmoles of cholesterol solubilized per mmole of added phospholipid was calculated by the method of least squares. Approximately the same quan-
3
HUMAN BILE LECITHIN
EGG LECITHIN (0.37i.02)
ITHIN (0.20f.01)
0
10
20
30
40
50
60
PHOSPHOLIPID (mmoles/liter)
Fig. I. Cholesterol solubilization by different concentrations of egg (A-A), cabbage (O-O) and bile (O-O) lecithins and egg (0-n) and cabbage (m-m) lysolecithins added to 180 mM sodium taurocholate. Crystalline cholesterol was incubated with the solutions for 6 days at 37 “C.Insoluble material was then removed with a 0.22 pm Millipore filter. The figures in parentheses represent the regression coefficients + S.D. of the regression coefficient of the lines and were calculated by the method of least squares. B&him.
Biophys.
Acta, 270 (1972) 407-413
SOLUBILIZATION
4x=
OF CHOLESTEROL
tity of cholesterol was solubilized by each of the lecithins when added to a 180 mM sodium taurocholate solution (Fig.) even though the egg, human bile and cabbage leaf lecithins differed markedly in their fatty acid composition. For every mmole of egg licithin added to a bile salt solution, 0.37 & 0.02 mmole of cholesterol was solubilized; for human bile lecithin 0.39 f 0.04 and for cabbage lecithin 0.35 rir:0.02 mmole Added cabbage and egg lysolecithins solubilized less cholesterol than their respective lecithins (Fig. I) but both lysolecithins solubilized approximately the same quantity of cholesterol. Cabbage lysolecethin, which contained 61 % unsaturated fatty accids, solubilized 0.20 & 0.01 mmole of cholesterol and egg lysolecithin which contained only saturated fatty acids solubilized 0.19 -& 0.01 mmole of cholesterol per mmole of added lysolecithin (Fig. I). E$ect of modification of cholesterol on its micellar solubilizatiolz. Cholestanol is a hydrogenated product of cholesterol and differs in that it does not have a double bond at CrCB. To test the effect this change in the structure of cholesterol has on its solub~zation, we measured the solub~zation of cholestanol by increasing concentrations of egg lecithin in 180 m&I solutions of sodium taurocholate. These solutions solubilized significantly smaller amounts of cholestanol than cholesterol (Fig. 2). One 30
CHOLESTERYL
5
0
10
ACETATE
20
30
LECITHIN
(mmol~r/lihc)
40
(0.04+.01)
50
60
Fig. 2. Cholesterol(o-o), cholestanol(&-A) and cholesterylacetate (O-O), solubilizationby differentconcentrationsof egg lecithin added to 180 mM sodiumtaurocholate.Conditionsof the experimentare the sameas in Fig. I. mmole of added lecithin solubilized 0.37 rfi.0.02 mmole of cholesterol but only 0.29 f 0.01 mmole of cholestanol. We also studied the solubility of cholesterol esters. The 180 mM solution of sodium taurocholate itself solubilized 1.2 mmole per 1 of cholesteryl acetate (Fig. 2). For every added mmole of lecithin, only 0.04 f o.or mmole of cholesteryl acetate was solubilized. Less than 0.1 mmole per 1 of cholesteryl oleate was solubilized by the bile %~%&a. Biophys. Acta,
270
(1972)
407-4~3
D. H.
412
NEIDERHISER,
H. P. ROTH
salt solution itself. No additional cholesteryl oleate was solubilized when lecithin was added to the bile salt solution. The solubilization of cholesterol, cholestanol and cholesteryl acetate was also tested by the method of Small d at .ao. r-ml aliquots of ethanolic solutions of 180 mM sodium taurocholate, 40 mM lecithin and 18 mM cholesterol or cholestanol or cholesteryl acetate were mixed, dried and the residue reconstituted with I ml of water. The solutions were allowed to equilibrate 20. After equilibration all of the cholesterol was in solution, however, only 14 mmole per 1 of cholestanol and 4 mmole per 1 of cholesteryl acetate was in solution; similar to the values reported above with our technique&. DISCUSSION
This study demonstrated that differences in the degree of unsaturation of lecithin had little effect on the quantity of cholesterol solubilized by a micellar solution of bile salts and lecithin. This suggests that differences in the degree of saturation of lecithin secreted in bile is unlikely to be important in the cause of gallstones in man. In the report of Blomstrand and Ekdahl& the fatty acid composition of lecithins from most patients with gallstones was within the range of fatty acid compositions recently reported in normal bilezf. The onlv abnormal samples were from z patients with occlusion of the cystic duct. In these biles, there tended to be less unsaturated fatty acids in the lecithins, but changes in the bile secondary to the occlusion itself could not be ruled out. The cabbage lecithin used in this study contained 830/ounsaturated fatty acids and 49% of the total fatty acids were linolenic acid (C,,,,) (Table I). When compared with egg and human bile lecithins which only contain 5o:/, unsaturated fatty acids and no linolenic acid (Table I), no difference in the cholesterol solubilized by the added lecithins was found (Fig. I). Hegardt and Dams2 produced an egg lecithin with increased unsaturated fatty acids by feeding chickens safflower oil. They also found no effect of the degree of unsaturation of the fatty acids of lecithin on cholesterol solubilization. Varying quantities of aracl~idollic acid (C,,,,) are found in human bile lecithin. However, neither our studies nor those of Hegardt and Dam2z or Saunders and Well9 have tested the possible solubilizing powers of lecithins containing major quantities of arachidonic acid. Since the quantity of this fatty acid in human bile is normally small (approximately 5 76 of the total fatty acids, Table I), it may not be important. When added to a bile salt solution, lysolecithin with one fatty acid solubilized only half as much cholesterol as did lecithin with z fatty acids (Fig. I). This suggested that the fatty acid was critical for the solubilization of cholesterol by these phospholipids. We anticipated that if the nature of the fatty acid of ~hos~Ilolipid was important, it should be most evident in our studies with Iysolecithin. Egg lysolecithin, like human bile lysolecithin, has no unsaturated fatty acids. It was possible to compare this saturated lysolecithin with the lysolecithin of cabbage which has 61~& unsaturated fatty acids. No difference in the cholesterol solubilizing powers of these lysolecithins, when added to a bile salt solution, was observed. This finding supports the concept that moderate changes in the saturation of fatty acids do not alter cholesterol solubilization by phospholipids. While changes in the fatty acids of the ~l~ospholi~id had no effect on cholesterol Biockim. Biopkys. z‘fcta, 270 (1972)
407-413
413
SOLUBILIZATION OF CHOLESTEROL
solubilization, motications of cholesterol did effect solubilization. Hydrogenation of the double bond of cholesterol decreased the so~ub~~at~on slightly but s~~i~~ant~~. There are several possible explanations, among them is a possible effect on the reactivity of the hydroxyl group. The possible significance of the hydroxyl group is also suggested by the diminished solubilization when esters are formed. Cholesteryl acetate was much less soluble than was cholesterol. Cholesteryl oleate was essentially insduble but here a long chain fatty acid had been added and marked changes would be expected. Significant quantities of cholesterol esters have not been demonstrated in bile. However, Nakayama@ has found a small amount of cholesterol esters in gallstones. The insolubility of the esters of cholesterol in mode1 bile solutions as observed in this study fits with their absence from bile and their presence in gallstones. ACKNOWLEDGEMENT
The authors thank Dr Ralph De Palma who provided the samples of human gallbladder bile. REFERENCES 1 2 3 4 5 6 7 8 g IQ IL IZ
13 r4 15 16
17 18 19 20 21 22
D. H. Neiderhiser and H. P. Roth, J. Lab. Clin. Med., 74 (1969) 493. A. F. Hofmann, G&roentemZogy, 48 (1965) 484” W. H. Admirand and D. M. Small, J. Clin. .i%vest., 47 (1968) 1043. D. H. Neiderhiser and H. P. Roth, Prac, Sac. &q%l. Bicrl. Med., 128 (1968) 221. R. Blomstrand and P. Ekdahl, Proc. Sot. Exptt. BioE. Med., x04 (1960) 205. D. R. Saunders and M. A. Wells, Be’oclrd%.Biopkys. A&a, 176 { fg6g) 828. A. Gottfries, S. Nilsson, B. Samuelsson and T. Schersttk, Scud. J. C&k La&. fwesd., z1{1g68j 168. 3. Borgstr&m, Acta C&em. Sand., II (~957) 749. F. Nakayama, J. Lab. Ctin. Med., 73 (1969) 623. D. H. Neiderhiser, H. P. Roth and L. T. Webster, J. Lab. CZilp. Med., 68 (1966) go. L. W. Wheeldon, J. Lipid Res., I (x960) 439, D. J. Hanahan, J. Biol. Ckem., 195 (rgp) 199. L. F. Fieser and M. Fieser, Steroids, Reinhold, New York, 1959, p. 28. R. D. Bennett and E. Heftmann, J. Ckromatog., 9 (1962) 359. A. H. Blat& Urga& Synbhssis, Wiley, New York, x943, p. 193. Il. Kritchevsky, D. S. Martak and G. H, Rothblat, Aarsl. B&&em., 5 (~963) 388. W. W. Wells and C, A. Baumann, A+x%. Biochem. Bi5#qs., 53 (1954) 471. G. V. Vahouny, C, R. Borta, R. M. Mayer and C. R. Treadwell, A%&. B&&e%, I (1960) 371. D. H. Neiderdiser and E-I.P. Roth, GasCroen&xaEogy,58 (1970) 26. D. M. Small, M, C. BourgBs and D. G. Dervichian, Biochim. Bio@ys. Acta, 125 (1966) 563. J. A. Balint, E. C. Kyriakides, H. L. Spitzer and E. S. Morrison, J. Lipid Res., 6 (1965) 96. F. G. Hegardt and H. Dam, Emahrungswissen, xo (x971) 223. Biochim. Bioj5bys.
Acta,
270 (1972) 407-4~3