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Solubility of cholesterol in vitro promoted by oxidation products of cholesterol
Atherosclerosis, 43 (I 982) 95 104 Elsevier/North-Holland Scientific Publishers.
95 Ltd
Solubility of Cholesterol In Vitro Promoted by Oxidation Products of Cholesterol L.H.
Krut
Depurtment of Medicine. Barugwunuth Hospital. und the lJkwrsit_v of the Witwutersrund, 2013, Johannesburg (South Africu)
P. 0. Bertshum
(Received I October, 198 I ) (Revised, received I November, 198 I ) (Accepted 13 November, I98 I)
Summary Compounds promoting the solubility of cholesterol could have a role in preventing its crystallisation and accumulation in tissue and in body fluids. A small quantity of phosphatidylcholine added to a supersaturated solution of cholesterol in a triglyceride oil has limited capacity to maintain solubility of cholesterol. Small quantities of oxidation products of cholesterol (oxysterols) have no material effect on the solubility of cholesterol in this system. However, the combination of phosphatidylcholine and oxysterols effectively maintains cholesterol in stable solution. In aqueous medium, the capacity of phosphatidylcholine to solubilise a molar excess of cholesterol is greatly increased by oxysterols. Oxidation products of cholesterol and phosphatidylcholine acting synergistically enhance enormously the solubility of cholesterol both in supersaturated solution in a triglyceride oil and in aqueous medium. Key words:
Cholesterol companions
- Micellar solution - Phospholipid
Introduction It was shown some time ago [l] that the crystallisation rate of cholesterol from supersaturated solution in a triglyceride oil is decreased by the addition of small quantities of lipids extracted from human serum. The addition of larger quantities of This work was supported
0021-9150/82/OOOC-0000/$02.75
by grants
from the University
of the Witwatersrand.
c 1982 Elsevier/North-Holland
Scientific
Publishers,
Ltd.
96
serum lipids further decreased crystallisation rate or prevents crystallisation. Fractionation of the serum lipids into polar and nonpolar fractions showed, inter alia, that the substances promoting solubility of cholesterol are contained entirely in the polar fraction, namely, the phosphatides, and, of these, only phosphatidylcholine has a significant effect. It was subsequently observed [2] that the capacity of serum phosphatides to promote the solubility of cholesterol in this system is dependent on the presence of impurities in the reagent cholesterol. The relevant impurities appeared to be oxidation products of cholesterol but this observation was not elucidated further at that time. This report describes the effect of certain oxidation products of cholesterol on the solubility of cholesterol in supersaturated solution in a triglyceride oil. Observations on the effect of these compounds on the solubility of cholesterol in an aqueous medium are also described. A part of this work was reported at a meeting of the Society for Endocrinology, Metabolism and Diabetes of Southern Africa [3].
Methods Pure phosphatidylcholine was isolated from hens egg yolk, made up to volume in ethanol, its concentration determined and the solution stored as described previously
141. Oxidation products of cholesterol were formed from chromatographically pure cholesterol (Merck) by the method of Bergstrom and Wintersteiner [5,6] yielding a mixture of cholesterol and a number of its oxidation products (oxysterol mixture). The sterols were separated by silicic acid thin layer chromatography (TLC) on Kieselgel HF 254 (Merck) and developed in the solvent system ethyl acetate/benzene/acetic acid in the ratios 75 : 25 : 1 by volume. The crystallisation of cholesterol from supersaturated solution was studied as solutions of cholesterol described previously [ 11. For these studies the supersaturated (crystallisation mixture) were made up with cholesterol in the amounts given below, and adding sunflower seed oil, containing 3.5% free fatty acids from this oil, to a combined weight of 25 g and heated in an oven at 90°C to bring the cholesterol into solution. Where the oxysterol mixture was included in the system, a weighed amount in powder form, as given below, was added to the pure cholesterol, the oil then added to a combined weight of 25 g and the sterols dissolved as above. 2.5 ml aliquots of the hot crystallisation mixture, both with and without oxysterol mixture, were pipetted into cuvettes, brought to 90°C for 10 min, the contents thoroughly mixed on a whirlimixer, and placed in a water bath at 25’C. The crystallisation of cholesterol was measured by the change in absorbance of the solution [I]. Where phosphatidylcholine and/or oxysterol reference compounds (purchased from Steraloids Inc., New York) were introduced into the system, known amounts dissolved in ethanol were pipetted into cuvettes, the solvent evaporated under nitrogen, the hot crystallisation mixture added, thoroughly mixed, heated and crystallisation at 25°C monitored as above.
97
Solutions in organic solvents of cholesterol and phosphatidylcholine and of oxysterol mixture, phosphatidylcholine and cholesterol, in the amounts given below, were freed of solvent under nitrogen. To this was added saline (I55 mM NaCl) mixed on a whirlimixer and a drop of the preparation examined under the microscope through crossed polarisers. The above lipid mixtures were also dissolved in chloroform, a drop allowed to dry on a microscope slide, covered with saline and examined as above.
Results The oxysterol mixture Oxidation of cholesterol by the method of Bergstrom and Wintersteiner [5,6] yields the products 7a-hydroxycholesterol, 7,&hydroxycholesterol, 7-ketocholesterol and A6-cholestene-3/3,5cw-diol. This method requires that cholesterol dissolved in hot ethanol is added to an alkaline solution of sodium stearate and air bubbled through the sol for 5 h. Bubbling oxygen through the sol gave a substantially higher yield of the same oxidation products that were obtained with air. The product obtained with oxygen was used for these studies. After oxidation the sol is acidified and the lipids extracted in diethyl ether. Stearic acid can be removed by saponification but this step was omitted. The final product (oxysterol mixture) consequently contained stearic acid. Figure 1 shows a TLC plate of pure cholesterol, oxysterol reference compounds and the oxysterol mixture after spraying with 50% sulphuric acid and heating in an oven at 130°C until the organic material is completely charred. The two 7-hydroxy compounds can also be identified by their characteristic Lifshtitz reaction [see 7, References]. The most polar. constituent of the oxysterol mixture, for which no reference compound was available, is presumably A6-cholestene-3P,Sa-diol [6,7]. TLC of the oxysterol mixture on microscope slides coated with Kieselgel gave a rough measure of the relative proportions of its constituents by scanning the charred preparation on a Gelman densitometer ACD 15. The proportions were: cholesterol 50.1%; stearic acid 20.6%; 7-ketocholesterol 7.7%; 7a-hydroxycholesterol 9.7%; 7fi-hydroxycholesterol 9.8%; A6-cholestene-3/3,5a-diol (presumed) 2.2%. Crystallisation of cholesterol from supersaturated solution in oil Figure2 shows the change with time in the optical density of the crystallisation mixture containing: (i) cholesterol, (ii) cholesterol plus phosphatidylcholine, (iii) cholesterol plus oxysterol mixture and (iv) cholesterol plus oxysterol mixture plus phosphatidylcholine. It can be seen that only the combination of phosphatidylcholine and oxysterol mixture effectively prevents crystallisation of cholesterol from supersaturated solution. The amount of oxysterol mixture added to these systems was established by trial and error and is about the least amount found to keep cholesterol dissolved in supersaturated solution when combined with the stated quantity of phosphatidylcholine. Figure 3 shows the above preparations 7 days after crystallisation is complete. It is
Fig. I. Thin layer chromatography of reference compounds and the oxysterol mixture. From left to right: cholesterol, 7-ketocholesterol, 7/3-hydroxycholesterol (note contamination with ‘I-ketocholesterol), 7ahydroxycholesterol and oxysterol mixture. Compounds in the oxysterol mixture from above downwards are cholesterol, stearic acid, 7-ketocholesterol. 7jShydroxycholestero1, 7a-hydroxycholesterol and Ahcholestene-3j3, 51x-dial (presumed).
usual for the cholesterol in the preparation containing oxysterol mixture plus phosphatidylcholine to remain in stable solution for several weeks when stored in the dark at room temperature. No crystals are seen microscopically through crossed polarisers in this preparation. Figure4 shows the effect on crystallisation of cholesterol in systems to which oxysterol reference compounds have been added. These compounds alone also have no demonstrable effect on the solubility of cholesterol. The addition to phosphatidylcholine of either 7-ketocholesterol or 2Shydroxycholesterol confers little additional solubilising action. The effect of 7a-hydroxycholesterol is substantial and that of 7/?-hydroxycholesterol considerably greater still. Of the compounds tested, those
C
Chdesterd t Oxysterol Mixture
0
L IO
&7+-F----20
30
40
50
0
IO
20
30
40
50
60
Mmutes
Fig 2. Change in absorbance (O.D.) with time in supersaturated solutions of cholesterol in oil. The crystallisation mixture was made up with I.575 g cholesterol. Where oxysterol mixture was added to the crystalhsation mixture, this was in the amount of 5X mg. The graphs on the left show change in O.D. of cholesterol alone (C) and when 3.75 mg phosphatidylcholine (tlecithin) is added to this. The graphs on the right show changes in O.D. when the crystallisation mixture contains oxysterol mixture (C) and when 3.75 mg phosphatidylcholine is added to this. Oxysterols alone have no material effect on crystallisation but the addition of phosphatidylchohne to this prevents the crystallisation of cholesterol.
Fig. 3. Preparations as for Fig. 2 stored in the dark at room temperature for 7 days. The tu bes from left to right are cholesterol alone, cholesterol plus phosphatidylcholine, cholesterol plus oxystet .ol mixture and the latter plus phosphatidylcholine; the last showing no evidence of cholesterol crystallisa
.8.7+Pc+7-KETO
6-
+Pc+25-
OH
CXY,5E .q_ 6 .g 0” .3_
.2I-
+ PC +7U - OH+7P _
IO
26
5b
so
70
86
lb0
- OH
Ii0
Minutes Fig. 4. The effect on crystallisation of cholesterol of different oxidation products of cholesterol. The crystallisation mixture was made up with I.40 g cholesterol and to each tube (2.5 ml) was added I X6 mg phosphatidylcholine (PC) and 5.0 mg each of 25-hydroxycholesterol (25-OH) or 7-ketocholesterol (7-keto), or 7a-hydroxycholesterol(7~OH) or 7/?-hydroxycholestero1(7/3-OH) or both 7o-OH and 78-OH.
Fig. 5. Cholesterol plus phosphatidylcholine and cholesterol plus oxysterol mixture plus phosphatidylcholine dispersed in saline. Lipids remain deposited in the former and are completely dispersed in the latter. See text for details.
101
with an additional hydroxyl group on the sterol nucleus have much the greater effect on solubilising cholesterol. The amount of stearic acid added to each cuvette with oxysterol mixture is about 1 mg, in a system already containing about 78 mg of free fatty acids from sunflower seed oil. Small quantities of additional fatty acids do not affect the solubility of cholesterol in this system [l]. Adding 10 mg of stearic acid to these systems has no demonstrable further effect on the solubility of cholesterol. Studies on cholesterol solubilisation in saline The effect of oxysterols on the solubilisation of cholesterol in saline was demonstrated crudely in two ways. Phosphatidylcholine (1.86 mg) was added to (i) 2.0 mg cholesterol, and (ii) 2.0 mg oxysterol mixture plus 1.0 mg cholesterol. These preparations were freed of solvent in a stream of nitrogen, 1.0 ml saline was added to each and mixed on a whirlimixer. Preparation (i) showed some opalescence, but with what seems to be the bulk of the lipid still deposited at the bottom of the tube. In preparation (ii) opalescence was more marked with all the lipid dispersed in the saline (Fig. 5). The aqueous phase of preparation (i) showed many cholesterol crystals and large clumps of birefringent crystalline material (Fig. 6). In preparation (ii) the only birefringent material is an
Fig. 6. The aqueous phase of the tube on the left in Fig. 5 viewed polarisers, showing abundant cholesterol crystals. X 183.
microscopically
through
crossed
Fig. 7. The aqueous phase of the tube on the right in Fig. 5 viewed microscopically polarisers, showing occasional liquid crystals and no cholesterol crystals. X 183.
through
crossed
Fig. 8. Cholesterol plus oxysterol mixture plus and viewed microscopically through crossed birefringent crystalline material which goes formation of some liquid crystals. See text for
phosphatidylcholine in chloroform left to dry on a slide polarisers (X 183). In the dry state (at left) there is into solution when covered with saline (at right) with details.
occasional spherular or tubular liquid crystal (Fig. 7) of the type seen when phosphatidylcholine alone is dispersed in this fashion. Since the molar ratio of phosphatidylcholine to cholesterol in preparation (i) was far less than that required to micellise all the cholesterol, the presence of crystalline cholesterol was to be expected [4]. In preparation (ii) with the same content of pure cholesterol as in (i), all the sterols seem to have been solubilised. The lipid mixtures in preparations (i) and (ii) were taken up in 1.0 ml chloroform and a few drops of each left to dry on a microscope slide. Saline run under a cover slip produced little change in preparation (i). In preparation (ii) there is a rapid disappearance of birefringent material with formation of some spherular and tubular liquid crystals (Fig. 8).
Discussion In the original observations on supersaturated solutions of cholesterol in oil [l] we used British Drug Houses (B.D.H.) ‘Laboratory Reagent’ Cholesterol. Subsequently B.D.H. ‘Biochemical Reagent’ Cholesterol was used but with this material the usual amount of phospholipid did not prevent cholesterol crystallisation. It was at that
103
time established from B.D.H. that sheep wool fat had been the source material for Laboratory Reagent Cholesterol and beef spinal cord the source material for Biochemical Reagent Cholesterol. TLC of Laboratory Reagent Cholesterol and the sterol fraction isolated from sheep wool fat (obtained from local sources) showed both to contain about 10 additional compounds, including some of those formed for the present studies. It is well known that cholesterol readily undergoes oxidation when exposed to light and air [7,8]. The oxidised compounds of cholesterol found in sheep wool fat presumably form through such exposure and could account for their presence in impure Laboratory Reagent Cholesterol. The Biochemical Reagent Cholesterol was a much purer preparation and only when it was subjected to oxidation did the addition of phospholipid promote solubility of cholesterol [2]. Attempts at that time to isolate the compound in sheep wool fat and in Laboratory Reagent Cholesterol which together with phospholipid promoted solubility of cholesterol indicated that numerous compounds had this property (unpublished). It seems probable that the oxysterols used in the present studies are not the only cholesterol companions with this action. The effect of phosphatidylcholine alone in promoting solubility of cholesterol in supersaturated solution in oil, in the amounts used, is limited. The addition of oxidation products of cholesterol of the order of 1% of the total sterols, which alone have little effect on increasing the solubility of cholesterol, enhance enormously the solubility of cholesterol when phosphatidylcholine is present. There would therefore seem to be synergism between these classes of compounds in promoting the solubility of cholesterol and maintaining its solubility in supersaturated solution. In aqueous medium phospholipids emulsify triglycerides and cholesterol esters and micellise cholesterol [4,9,10]. Effective micellisation of cholesterol requires that cholesterol and phosphatidylcholine are present in equimolar proportions and the preparation subjected to prolonged sonication [4]. In striking contrast, the presence of certain oxysterols allows phosphatidylcholine to ‘solubilise’, presumably in micellar form, a substantial molar excess of cholesterol with relatively minor physical encouragement (Figs. 5,6 and 7) or none at all (Fig. 8). Oxysterols would therefore also seem to act synergistically with phosphatidylcholine in promoting the solubility of cholesterol in aqueous medium. The oxysterols most effective in promoting solubility of cholesterol are those with an additional hydroxyl group on the nucleus of the cholesterol molecule (Fig. 4). This calls to mind one of the changes to cholesterol when bile acids are synthesized with bile acids solubilise cholesterol in bile [ 121. Unconfrom it [ 111. Phospholipids jugated di- and tri-hydroxy bile acids also promote the solubility of cholesterol in supersaturated solution in a triglyceride oil when phosphatidylcholine is present [ 131. Conceivably oxysterols promote solubility of cholesterol in a manner similar to that by which bile acids promote the solubility of cholesterol in bile. It seems possible that oxidation products of cholesterol which promote the solubility of cholesterol in vitro could have biological relevance in a number of ways. A study to examine one such possibility is the subject of another communication 1141.
104
References I Wilkens, J.A. and Krut, L.H., Stabilization of supersaturated extracts - A new serum parameter associated with ischaemic (1963) 15.
cholesterol solutions by serum lipid heart disease, J. Atheroscler. Res., 3
2 Wilkens, J.A. and Krut, L.H., The effect of glucose on the crystallisation Res., 5 (1965) 516.
of cholesterol,
J. Atheroscler.
3 Krut, L.H., Effect of certain carbohydrates and sterols on the capacity of phosphatidylcholine to solubilize cholesterol, Abstract S. Afr. J. Sci., Suppl. 73 (1977) i. 4 Horwitz, C., Krut, L.H. and Kaminsky, L., Cholesterol uptake by egg-yolk phosphatidylcholine. B&him. Biophys. Acta, 239 (I 971) 329. 5 Bergstrom, S. and Wintersteiner, 0.. Autoxidation of sterols in colloidal aqueous solution - The nature of the products formed from cholesterol, J. Biol. Chem., 141 (1941) 597. 6 Bergstrom, S. and Wintersteiner. 0.. Autoxidation of cholesterol in colloidal aqueous solution, Part 2 (d-Cholestenediol-3P,5, a rearrangement product of 7 (/3)-hydroxycholesterol), J. Biol. Chem., 143 (I 942) 503. 7 Fieser, L.F. and Fieser, M., Steroids, Reinhold, New York, 1959, p. 233. 8 Hais, I.M. and Myant, N.B., Photolysis of cholesterol during biological experiments, Biochem. J., 94 (1965) 80. 9 Horwitz, C., Krut, L.H. and Kaminsky, L.S., Some properties of particles of egg-yolk phosphatidylcholine and cholesterol, Chem. Phys. Lipids, 8 (1972) 185. 10 Horwitz, C., Krut, L.H. and Kaminsky, L.S., The emulsifying properties of egg-yolk phosphatidylcholine, Lipids, 7 (1972) 234. 1I Bjorkhem, I., Reaction mechanisms in bile acid synthesis. In: P. Back and W. Gerok (Eds.). Bile Acids in Human Diseases, F.K. Schattauer Verlag, Stuttgart, 1972. p. 9. 12 Admirand, W.H. and Small, D.M., Physicochemical basis of cholesterol gallstone formation in man, J. Clin. Invest., 47 (1968) 1043. 13 Krut, L.H., Postulated role for glucose in the genesis of cholesterol gallstones (Abstr.), S. Afr. J. Sci., Suppl. 74 (1978) x. 14 Krut, L.H., Clearance of subcutaneous implants of cholesterol in the rat promoted by oxidation products of cholesterol - A postulated role for oxysterols in preventing atherosclerosis, Atherosclerosis, 43 (1982) 105.
95 Ltd
Solubility of Cholesterol In Vitro Promoted by Oxidation Products of Cholesterol L.H.
Krut
Depurtment of Medicine. Barugwunuth Hospital. und the lJkwrsit_v of the Witwutersrund, 2013, Johannesburg (South Africu)
P. 0. Bertshum
(Received I October, 198 I ) (Revised, received I November, 198 I ) (Accepted 13 November, I98 I)
Summary Compounds promoting the solubility of cholesterol could have a role in preventing its crystallisation and accumulation in tissue and in body fluids. A small quantity of phosphatidylcholine added to a supersaturated solution of cholesterol in a triglyceride oil has limited capacity to maintain solubility of cholesterol. Small quantities of oxidation products of cholesterol (oxysterols) have no material effect on the solubility of cholesterol in this system. However, the combination of phosphatidylcholine and oxysterols effectively maintains cholesterol in stable solution. In aqueous medium, the capacity of phosphatidylcholine to solubilise a molar excess of cholesterol is greatly increased by oxysterols. Oxidation products of cholesterol and phosphatidylcholine acting synergistically enhance enormously the solubility of cholesterol both in supersaturated solution in a triglyceride oil and in aqueous medium. Key words:
Cholesterol companions
- Micellar solution - Phospholipid
Introduction It was shown some time ago [l] that the crystallisation rate of cholesterol from supersaturated solution in a triglyceride oil is decreased by the addition of small quantities of lipids extracted from human serum. The addition of larger quantities of This work was supported
0021-9150/82/OOOC-0000/$02.75
by grants
from the University
of the Witwatersrand.
c 1982 Elsevier/North-Holland
Scientific
Publishers,
Ltd.
96
serum lipids further decreased crystallisation rate or prevents crystallisation. Fractionation of the serum lipids into polar and nonpolar fractions showed, inter alia, that the substances promoting solubility of cholesterol are contained entirely in the polar fraction, namely, the phosphatides, and, of these, only phosphatidylcholine has a significant effect. It was subsequently observed [2] that the capacity of serum phosphatides to promote the solubility of cholesterol in this system is dependent on the presence of impurities in the reagent cholesterol. The relevant impurities appeared to be oxidation products of cholesterol but this observation was not elucidated further at that time. This report describes the effect of certain oxidation products of cholesterol on the solubility of cholesterol in supersaturated solution in a triglyceride oil. Observations on the effect of these compounds on the solubility of cholesterol in an aqueous medium are also described. A part of this work was reported at a meeting of the Society for Endocrinology, Metabolism and Diabetes of Southern Africa [3].
Methods Pure phosphatidylcholine was isolated from hens egg yolk, made up to volume in ethanol, its concentration determined and the solution stored as described previously
141. Oxidation products of cholesterol were formed from chromatographically pure cholesterol (Merck) by the method of Bergstrom and Wintersteiner [5,6] yielding a mixture of cholesterol and a number of its oxidation products (oxysterol mixture). The sterols were separated by silicic acid thin layer chromatography (TLC) on Kieselgel HF 254 (Merck) and developed in the solvent system ethyl acetate/benzene/acetic acid in the ratios 75 : 25 : 1 by volume. The crystallisation of cholesterol from supersaturated solution was studied as solutions of cholesterol described previously [ 11. For these studies the supersaturated (crystallisation mixture) were made up with cholesterol in the amounts given below, and adding sunflower seed oil, containing 3.5% free fatty acids from this oil, to a combined weight of 25 g and heated in an oven at 90°C to bring the cholesterol into solution. Where the oxysterol mixture was included in the system, a weighed amount in powder form, as given below, was added to the pure cholesterol, the oil then added to a combined weight of 25 g and the sterols dissolved as above. 2.5 ml aliquots of the hot crystallisation mixture, both with and without oxysterol mixture, were pipetted into cuvettes, brought to 90°C for 10 min, the contents thoroughly mixed on a whirlimixer, and placed in a water bath at 25’C. The crystallisation of cholesterol was measured by the change in absorbance of the solution [I]. Where phosphatidylcholine and/or oxysterol reference compounds (purchased from Steraloids Inc., New York) were introduced into the system, known amounts dissolved in ethanol were pipetted into cuvettes, the solvent evaporated under nitrogen, the hot crystallisation mixture added, thoroughly mixed, heated and crystallisation at 25°C monitored as above.
97
Solutions in organic solvents of cholesterol and phosphatidylcholine and of oxysterol mixture, phosphatidylcholine and cholesterol, in the amounts given below, were freed of solvent under nitrogen. To this was added saline (I55 mM NaCl) mixed on a whirlimixer and a drop of the preparation examined under the microscope through crossed polarisers. The above lipid mixtures were also dissolved in chloroform, a drop allowed to dry on a microscope slide, covered with saline and examined as above.
Results The oxysterol mixture Oxidation of cholesterol by the method of Bergstrom and Wintersteiner [5,6] yields the products 7a-hydroxycholesterol, 7,&hydroxycholesterol, 7-ketocholesterol and A6-cholestene-3/3,5cw-diol. This method requires that cholesterol dissolved in hot ethanol is added to an alkaline solution of sodium stearate and air bubbled through the sol for 5 h. Bubbling oxygen through the sol gave a substantially higher yield of the same oxidation products that were obtained with air. The product obtained with oxygen was used for these studies. After oxidation the sol is acidified and the lipids extracted in diethyl ether. Stearic acid can be removed by saponification but this step was omitted. The final product (oxysterol mixture) consequently contained stearic acid. Figure 1 shows a TLC plate of pure cholesterol, oxysterol reference compounds and the oxysterol mixture after spraying with 50% sulphuric acid and heating in an oven at 130°C until the organic material is completely charred. The two 7-hydroxy compounds can also be identified by their characteristic Lifshtitz reaction [see 7, References]. The most polar. constituent of the oxysterol mixture, for which no reference compound was available, is presumably A6-cholestene-3P,Sa-diol [6,7]. TLC of the oxysterol mixture on microscope slides coated with Kieselgel gave a rough measure of the relative proportions of its constituents by scanning the charred preparation on a Gelman densitometer ACD 15. The proportions were: cholesterol 50.1%; stearic acid 20.6%; 7-ketocholesterol 7.7%; 7a-hydroxycholesterol 9.7%; 7fi-hydroxycholesterol 9.8%; A6-cholestene-3/3,5a-diol (presumed) 2.2%. Crystallisation of cholesterol from supersaturated solution in oil Figure2 shows the change with time in the optical density of the crystallisation mixture containing: (i) cholesterol, (ii) cholesterol plus phosphatidylcholine, (iii) cholesterol plus oxysterol mixture and (iv) cholesterol plus oxysterol mixture plus phosphatidylcholine. It can be seen that only the combination of phosphatidylcholine and oxysterol mixture effectively prevents crystallisation of cholesterol from supersaturated solution. The amount of oxysterol mixture added to these systems was established by trial and error and is about the least amount found to keep cholesterol dissolved in supersaturated solution when combined with the stated quantity of phosphatidylcholine. Figure 3 shows the above preparations 7 days after crystallisation is complete. It is
Fig. I. Thin layer chromatography of reference compounds and the oxysterol mixture. From left to right: cholesterol, 7-ketocholesterol, 7/3-hydroxycholesterol (note contamination with ‘I-ketocholesterol), 7ahydroxycholesterol and oxysterol mixture. Compounds in the oxysterol mixture from above downwards are cholesterol, stearic acid, 7-ketocholesterol. 7jShydroxycholestero1, 7a-hydroxycholesterol and Ahcholestene-3j3, 51x-dial (presumed).
usual for the cholesterol in the preparation containing oxysterol mixture plus phosphatidylcholine to remain in stable solution for several weeks when stored in the dark at room temperature. No crystals are seen microscopically through crossed polarisers in this preparation. Figure4 shows the effect on crystallisation of cholesterol in systems to which oxysterol reference compounds have been added. These compounds alone also have no demonstrable effect on the solubility of cholesterol. The addition to phosphatidylcholine of either 7-ketocholesterol or 2Shydroxycholesterol confers little additional solubilising action. The effect of 7a-hydroxycholesterol is substantial and that of 7/?-hydroxycholesterol considerably greater still. Of the compounds tested, those
C
Chdesterd t Oxysterol Mixture
0
L IO
&7+-F----20
30
40
50
0
IO
20
30
40
50
60
Mmutes
Fig 2. Change in absorbance (O.D.) with time in supersaturated solutions of cholesterol in oil. The crystallisation mixture was made up with I.575 g cholesterol. Where oxysterol mixture was added to the crystalhsation mixture, this was in the amount of 5X mg. The graphs on the left show change in O.D. of cholesterol alone (C) and when 3.75 mg phosphatidylcholine (tlecithin) is added to this. The graphs on the right show changes in O.D. when the crystallisation mixture contains oxysterol mixture (C) and when 3.75 mg phosphatidylcholine is added to this. Oxysterols alone have no material effect on crystallisation but the addition of phosphatidylchohne to this prevents the crystallisation of cholesterol.
Fig. 3. Preparations as for Fig. 2 stored in the dark at room temperature for 7 days. The tu bes from left to right are cholesterol alone, cholesterol plus phosphatidylcholine, cholesterol plus oxystet .ol mixture and the latter plus phosphatidylcholine; the last showing no evidence of cholesterol crystallisa
.8.7+Pc+7-KETO
6-
+Pc+25-
OH
CXY,5E .q_ 6 .g 0” .3_
.2I-
+ PC +7U - OH+7P _
IO
26
5b
so
70
86
lb0
- OH
Ii0
Minutes Fig. 4. The effect on crystallisation of cholesterol of different oxidation products of cholesterol. The crystallisation mixture was made up with I.40 g cholesterol and to each tube (2.5 ml) was added I X6 mg phosphatidylcholine (PC) and 5.0 mg each of 25-hydroxycholesterol (25-OH) or 7-ketocholesterol (7-keto), or 7a-hydroxycholesterol(7~OH) or 7/?-hydroxycholestero1(7/3-OH) or both 7o-OH and 78-OH.
Fig. 5. Cholesterol plus phosphatidylcholine and cholesterol plus oxysterol mixture plus phosphatidylcholine dispersed in saline. Lipids remain deposited in the former and are completely dispersed in the latter. See text for details.
101
with an additional hydroxyl group on the sterol nucleus have much the greater effect on solubilising cholesterol. The amount of stearic acid added to each cuvette with oxysterol mixture is about 1 mg, in a system already containing about 78 mg of free fatty acids from sunflower seed oil. Small quantities of additional fatty acids do not affect the solubility of cholesterol in this system [l]. Adding 10 mg of stearic acid to these systems has no demonstrable further effect on the solubility of cholesterol. Studies on cholesterol solubilisation in saline The effect of oxysterols on the solubilisation of cholesterol in saline was demonstrated crudely in two ways. Phosphatidylcholine (1.86 mg) was added to (i) 2.0 mg cholesterol, and (ii) 2.0 mg oxysterol mixture plus 1.0 mg cholesterol. These preparations were freed of solvent in a stream of nitrogen, 1.0 ml saline was added to each and mixed on a whirlimixer. Preparation (i) showed some opalescence, but with what seems to be the bulk of the lipid still deposited at the bottom of the tube. In preparation (ii) opalescence was more marked with all the lipid dispersed in the saline (Fig. 5). The aqueous phase of preparation (i) showed many cholesterol crystals and large clumps of birefringent crystalline material (Fig. 6). In preparation (ii) the only birefringent material is an
Fig. 6. The aqueous phase of the tube on the left in Fig. 5 viewed polarisers, showing abundant cholesterol crystals. X 183.
microscopically
through
crossed
Fig. 7. The aqueous phase of the tube on the right in Fig. 5 viewed microscopically polarisers, showing occasional liquid crystals and no cholesterol crystals. X 183.
through
crossed
Fig. 8. Cholesterol plus oxysterol mixture plus and viewed microscopically through crossed birefringent crystalline material which goes formation of some liquid crystals. See text for
phosphatidylcholine in chloroform left to dry on a slide polarisers (X 183). In the dry state (at left) there is into solution when covered with saline (at right) with details.
occasional spherular or tubular liquid crystal (Fig. 7) of the type seen when phosphatidylcholine alone is dispersed in this fashion. Since the molar ratio of phosphatidylcholine to cholesterol in preparation (i) was far less than that required to micellise all the cholesterol, the presence of crystalline cholesterol was to be expected [4]. In preparation (ii) with the same content of pure cholesterol as in (i), all the sterols seem to have been solubilised. The lipid mixtures in preparations (i) and (ii) were taken up in 1.0 ml chloroform and a few drops of each left to dry on a microscope slide. Saline run under a cover slip produced little change in preparation (i). In preparation (ii) there is a rapid disappearance of birefringent material with formation of some spherular and tubular liquid crystals (Fig. 8).
Discussion In the original observations on supersaturated solutions of cholesterol in oil [l] we used British Drug Houses (B.D.H.) ‘Laboratory Reagent’ Cholesterol. Subsequently B.D.H. ‘Biochemical Reagent’ Cholesterol was used but with this material the usual amount of phospholipid did not prevent cholesterol crystallisation. It was at that
103
time established from B.D.H. that sheep wool fat had been the source material for Laboratory Reagent Cholesterol and beef spinal cord the source material for Biochemical Reagent Cholesterol. TLC of Laboratory Reagent Cholesterol and the sterol fraction isolated from sheep wool fat (obtained from local sources) showed both to contain about 10 additional compounds, including some of those formed for the present studies. It is well known that cholesterol readily undergoes oxidation when exposed to light and air [7,8]. The oxidised compounds of cholesterol found in sheep wool fat presumably form through such exposure and could account for their presence in impure Laboratory Reagent Cholesterol. The Biochemical Reagent Cholesterol was a much purer preparation and only when it was subjected to oxidation did the addition of phospholipid promote solubility of cholesterol [2]. Attempts at that time to isolate the compound in sheep wool fat and in Laboratory Reagent Cholesterol which together with phospholipid promoted solubility of cholesterol indicated that numerous compounds had this property (unpublished). It seems probable that the oxysterols used in the present studies are not the only cholesterol companions with this action. The effect of phosphatidylcholine alone in promoting solubility of cholesterol in supersaturated solution in oil, in the amounts used, is limited. The addition of oxidation products of cholesterol of the order of 1% of the total sterols, which alone have little effect on increasing the solubility of cholesterol, enhance enormously the solubility of cholesterol when phosphatidylcholine is present. There would therefore seem to be synergism between these classes of compounds in promoting the solubility of cholesterol and maintaining its solubility in supersaturated solution. In aqueous medium phospholipids emulsify triglycerides and cholesterol esters and micellise cholesterol [4,9,10]. Effective micellisation of cholesterol requires that cholesterol and phosphatidylcholine are present in equimolar proportions and the preparation subjected to prolonged sonication [4]. In striking contrast, the presence of certain oxysterols allows phosphatidylcholine to ‘solubilise’, presumably in micellar form, a substantial molar excess of cholesterol with relatively minor physical encouragement (Figs. 5,6 and 7) or none at all (Fig. 8). Oxysterols would therefore also seem to act synergistically with phosphatidylcholine in promoting the solubility of cholesterol in aqueous medium. The oxysterols most effective in promoting solubility of cholesterol are those with an additional hydroxyl group on the nucleus of the cholesterol molecule (Fig. 4). This calls to mind one of the changes to cholesterol when bile acids are synthesized with bile acids solubilise cholesterol in bile [ 121. Unconfrom it [ 111. Phospholipids jugated di- and tri-hydroxy bile acids also promote the solubility of cholesterol in supersaturated solution in a triglyceride oil when phosphatidylcholine is present [ 131. Conceivably oxysterols promote solubility of cholesterol in a manner similar to that by which bile acids promote the solubility of cholesterol in bile. It seems possible that oxidation products of cholesterol which promote the solubility of cholesterol in vitro could have biological relevance in a number of ways. A study to examine one such possibility is the subject of another communication 1141.
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2 Wilkens, J.A. and Krut, L.H., The effect of glucose on the crystallisation Res., 5 (1965) 516.
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