Cholesterol: Complexes with metallic salts

Biochimica et Biophysics Acta, 337 (1974) 136-144 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 56381

CHOLESTEROL:

COMPLEXES WITH METALLIC

SALTS

P. MARFEY and H. CHESSIN Departments of Biological Sciences and Physics, State University of New York at Albany, Albany, N.Y. 12222 (U.S.A.)

(Received October I8th, 1973)

SUMMARY

Cholesterol forms complexes with CaCI, - 2Hz0 and with MgCl, - 6Hz0 consisting of two molecules of cholesterol and one molecule of inorganic salt. The two complexes have different solubilities in common organic solvents and are unstable in hydroxylic solvents, particularly in water. The reactivity of the A5 double bond of cholesterol with bromine is greater in the CaCl, * 2Hz0 complex than in the MgCl, * 6H,O complex, but the reverse is observed for acetylation of the 3j?-hydroxyl group of cholesterol in the two complexes. The infrared spectra of the two complexes display slight differences in the 400-1300 cm-l region. Lattice parameters of the two complexes

are similar to those of cholesterol.

The relative intensities

of the diffracted

powder lines are significantly different for the two complexes. The MgCl, - 6Hz0 complex shows considerably reduced intensities of the medium and strong lines relative to those of CaClz - 2H,O complex and cholesterol. It appears that the unit cell dimensions of cholesterol are not significantly changed upon complex formation with either salt, but that the positional parameters of the two complexes depart significantly from those of cholesterol.

INTRODUCTION

The first compound of cholesterol with a metallic salt, LiCl, was reported by Zwikker [I]. A more extensive research in this area was done by Hackmann [2] and by Garlinskaya [3] who have prepared a series of complexes of cholesterol with chloride salts of monovalent and divalent metal ions. Cholesterol is a major lipid constituent of biological membranes [4], and it is also found in the walls of the blood vessels where it may participate together with metallic salts in the development of arteriosclerotic placques [3]. The ion-binding property of cholesterol seemed to us sufficiently important to warrant further study of cholesterol-CaCl, and cholesterol-MgCl, complexes using chemical, infrared and X-ray powder diffraction techniques.

I37 MATERIALS

AND METHODS

Preparation

ofcomplexes

Both complexes were prepared according to the method of Hackmann [2] with minor modifications. For the preparation of Ca’+ complex, cholesterol (Matheson, Coleman and Bell, 5.82 g, 15 mmoles) was dissolved in IO ml of anhydrous acetone. CaCl, * 2H,O (I g, 6.8 mmoles) was dissolved in IO ml of 95 % ethanol. When the two solutions were mixed the precipitate was formed immediately. It was filtered next day, washed with anhydrous acetone and dried in the air. The yield was 5.9 g (94% of the theoretical yield). For the preparation of Mg”’ complex, cholesterol (4.22 g, 10.9 mmoles) was dissolved in 80 ml acetone. MgClz * 6Hz0 (I g, 4.93 mmoles) was dissolved in 7 ml of 95 % ethanol. Upon mixing the two solutions precipitate was formed which was filtered next day and washed with acetone. The yield of the air-dried product was 3.9 g (81% of the theoretical yield). Samples of the two complexes were decomposed with deionized water in the presence of ether. The liberated cholesterol was extracted into ether phase and was determined by the method of Zlatkis et al. [5]. The aqueous phase was analyzed for Ca’ + and Mg2+ using Perkin-Elmer Atomic Absorption Spectrometer Model 303. The emission line for Ca’+ at 2112 A and for Mg*+ at 2852 A was used for quantitation of the ions. The two complexes had the following composition: Anu~ysis: Calculated for (C,,H,,O), - CaCl, - zH,O: cholesterol, 84.03 ; Ca2+ , 4.36. Found: cholesterol, 81.10; Cazf, 4.21. Calculated for (C27H460)2 * MgCl, . 6H,O: cholesterol, 79.18 ; Mg*+, 2.49. Found: cholesterol, 87.30; Mg*+, 2.74. Solubility and stability of the metallic complexes

The approximate solubilities of the two complexes were determined in the following way. Weighed samples were placed in centrifuge tubes and shaken with a known volume of an organic solvent for I h at room temperature. After this time the contents were centrifuged, and the undissolved residues were dried and weighed. The difference in weights between the initial sample and a given residue was considered to represent the amount of the complexes solubilized in a given volume of a solvent. The stability of the two complexes in water was tested in the following way. zo-mg samples of the two complexes and of cholesterol control were partitioned between 8 ml ether and 8 ml deionized water. 2 ml of each phase was withdrawn for analysis after 5, 15 and 30 min from the start of the experiment. Ether phases were analyzed for cholesterol by the method of Zlatkis et al. [5] and the aqueous phases were analyzed for Ca* + and Mg*+ by the method of atomic absorption spectrometry as described above. The concentrations of the cholesterol and of the metal ions in the two respective phases was determined from the calibration curves determined with known standards. Bromination of the complexes

Two methods of bromination of the two cholesterol-metal ion complexes were used. In one, weighed solid samples of the two complexes and of cholesterol control were brominated in a saturated atmosphere of bromine vapor at room tem-

138

perature during 6 h under illumination from a desk lamp equipped with a 100-w GE bulb placed at a distance of approx. IO cm from the samples. The reaction vessel was cons~ucted from a glass-stoppered centrifuge tube. It contained a porous glass partition in the middle separating the sample from liquid bromine during the reaction. The brominated samples were dissolved in ether-water, and the ether phase containing brominated cholesterol was used for thin-layer chromatography. In the second method of bromination, zoo-mg samples of the complexes and of cholesterol control were suspended in 20 ml ether, and to each stirred suspension 0.1 ml of bromine was added. After r5 min, IO ml of the reaction mixture was removed, the excess bromine destroyed by the addition of solid Na,S03, and the mixture diluted with IO ml water. The second half of the reaction mixture was treated similarly after I h. Brominated cholesterol was extracted into ether, and the latter extract was used fot thin-layer chromatography. synthetic of cholesteroEgol,68-dibrom~~ and 5u,~~-dibromocholestane-3-o~e

The synthesis of cholesterol-5a,6/&dibromide was done according to the method of Windaus [6]. Briefly, I g of cholesterol in 10 ml ether was treated with 0.5 g bromine dissolved in 5 ml glacial acetic acid. The crystalline mass was filtered, crystals were washed with glacial acetic acid followed by water yielding I. I g of the product having m-p. 108-1 TO“C (ref. 7, I 12-114 “C). The conversion of cholesterol-5a,6~-dibromide to an isomeric cholesterol5P,6a-dibromide (m.p. 143 “C) was done in chloroform by the method of Barton and Miller [7]. The two isomeric dibromides were analyzed by thin-layer chromatography. The synthesis of ga,G@dibromocholestane-3-one was achieved by the method of Fieser [8]. From 0,529 of cholestero&ga,6j%dibromide 0.25 g of the product (first crop of crystals) having m.p. 73-74 “C (ref. 8, 73-75 “C) was isolated. Acetyhtion of the complexes

Samples (300 mg) of the two complexes and of cholesterol control were suspended in pyridine (IO ml) and treated with 2 ml of acetic anhydride at room temperature. z-ml sample of each reaction mixture was withdrawn after 0.5,2,6 and 20 h. The reaction in each sample was terminated by the addition of 2 ml water. The contents were further diluted with water, and the acetylated cholesterol extracted with ether. The crystalline products isolated from the water-washed ether extracts had m.p. I 14-116 “C (ref. 9, I 15 “C). The reaction products were analyzed by thin-layer chromatography. Thin-layer chromatography and.~~~rared spectra

Thin-layer chromatography was done with Baker-flex (flexible sheets) of Silica Gel xB-F, size 20 cm x 5 cm. The developing solvent was chloroform or cyclohexane-ethyl acetate (85 :IS, by vol.). The visualization of spots was done by exposure to iodine vapor. The spots of the dibromides were colored green after exposure to iodine vapor. Infrared spectra were obtained in KBr pellets (containing about I % of the two complexes or of cholesterol) with Perkin-Elmer Infrared Spectrometer Model 337 using air as the reference.

I39

X-ray pow&r diDaction

X-ray diffractometer diagrams of the two complexes and of cholesterol were taken employing a copper target X-ray tube, a nickel foil in front of the scintillation counter and a subsequent pulse he&t selection set to accept the copper Ka line. The crystalline powders were screened through a 325-mesh per inch sieve, sealed in a o.3-mm diameter quartz capillary, and the diagrams were taken in a I r4.59-mm diameter camera. Exposure times were from 20 to 24 h. Intensities from the films were measured by microdensitometering the three powder diagrams using a Joyc-Loebl densitometer to obtain values of the relative intensities. The films were indexed with the aid of a computer program on the basis of the triclinic system but no refinements on the parameters could be made since the quality of the diffraction lines did not warrant this. Application of the homogeneous axes and the Delaunay reduction techniques [IO] indicated that the pattern had been indexed on its smallest unit cell possible. RESULTS

The complexes formed between CaCl, - 2HzO or MgCI, * 6H20 and cholesterol consist of two molecules of cholesterol and one metal ion as indicated by the analysis for cholesterol and for the two metal ions (see Materials and Methods). The solubility of the complexes in common organic solvents is given in Table I. Both complexes are appreciably soluble (2.0 and 2.7 mg per ml) in such a non-polar solvent as benzene (E 2.28 at 20 “C). It is seen from Table I that solubility of the two complexes in several organic solvents is different. It is not proportional to the dielectric constant of the solvent and does not parallel the solubility of cholesterol. The relative stability of the two complexes was investigated in a biphasic water-ether system. The two-phase system was used because after dissociation of the complex the two components can readily be solubilized (cholesterol in ether and CaCl, or MgC12 in water). Samples (2 ml) of the two phases withdrawn after 5, 15 and 30 min of exposure to the biphasic solvent were analyzed for cholesterol and for the TABLE I APPROXIMATE

SOLUBILITIES

OF CHOLESTEROL-METALLIC

SALT COMPLEXES

Weighed samples of the complexes were placed in centrifuge tubes and shaken with a known volume of a solvent at room temperature. After I h the contents were centrifuged, the undissolved residues were dried and weighed. The difference in weights was taken to represent the amount of the complexes dissolved in a given volume of a solvent. Values for dielectric constants of the listed solvents were taken from refs II and 12. Solubility values for cholesterol in benzene and p-dioxane were taken from ref. 15. Solvent

Carbon tetrachloride Benzene Ether Chloroform Ethanol p-Dioxane

e at

20

2.23 2.28 4.33 5.05 24.00 2.21

“C

Solubilitv tmalmll CaCl, complex MgCla complex

Cholesterol

0.1

insoluble

321

2.0

2.7

I25

insoluble 3.7 1.8 insoluble

insoluble insoluble

357

2.0

insoluble

222

13 116

respective metal ion. The concentrations of free cholesterol and of the metal ions were approximately the same after 5, 15 and 30 min exposure to water-ether indicating that dissociation of the complexes into components had occurred before the first 5-min sample was withdrawn. The molar ratio of cholesterol to metal salt was close to 2 which is in good agreement with the analytical data obtained separately on the two complexes (see Materials and Methods). Only unchanged cholesterol was recovered from the two complexes and it was shown by thin-layer chromatography to be identical to the control sample of cholesterol. In chloroform it migrated with an R, value bf 0.400.42 and in cyclohexane-ethyl acetate (X5:15, by vol.) with an R, value between 0.28 and 0.30. When bromination was carried out with solid samples of the two complexes in a saturated bromine atmosphere under illumination with visible light no differential reactivity of the A5 double bond of cholesterol was observed. Cholesterol in both complexes and in the control sample was converted under these conditions to a brominated and oxidized compound, 5”,6P-dibromocholestane-3-one which was identical to the synthetic sample (m.p. 73-74 “C, RF (in chloroform) 0.91-0.95). A different reactivity of the A5 double bond of cholesterol toward bromine was observed with the two complexes when the bromination was carried out in ether at room temperature. The results, representing averages of two experiments, showing the relative abundance of various brominated products after 15 and 60 min of reaction are shown in Table 11. Cholesterol in the CaCl, complex appears to be more reactive toward bromine than in MgCl, complex. At the end of I h there was still observed a TABLE II BROMINATION OF CHOLESTEROL-CaQ AND CHOLESTEROL-MgCI*COMPLEXES IN ETHER Samples(200mg) of the two complexesand of cholesterolcontrol were suspendedin ether (20 ml) containing bromine (0.1ml), and the reaction mixtures were stirred at room temperature. Samples were withdrawnafter 15 and 60 min. The complexes were decomposed in water-ether, and the ether phase was analyzed by thin-layer chromatography. Reference compounds were run on the same plates to aid in identification of the spots. Average RF values (two experiments) of spots obtained with Baker-flex Silica Gel IB-F, 20 cm x 5 cm sheets, using chloroform as a solvent. The spots were classified as strong (s), medium (m) and weak (w). A medium and a weak spot contained approx. 40-60 % and 10-20 ‘A of the material of the strong spot, respectively. For identification of the reaction products the following reference compounds were used: I, 5a,6/?- or 5/3,6a-dibromocholestane-3-one; II, cholesterol-Q,6a-dibromide; III, cholesteroLga,68-dibromide; IV, cholesterol control. Sample

CaClz . 2Hz0 complex

MgClz . 6H20 complex

Cholesterol

RF of thin-layer

spots after

15 min

60 min

0.62 0.41 0.27 0.62 0.41 0.26 0.93 0.69 0.48 0.28

0.91 (m) 0.66 (s) 0.42 (s) 0.20 (w) o.7r (s) 0.49 (s) 0.23 (s) 0.89 (m) 0.62 (s) 0.44 (s)

(s) (s) (m) (s) (s) (s) (m) (s) (s) (w)

0.21

(w)

Identification

I II III IV II III IV I II III IV

141 TABLE III ACETYLATION OF CHOLESTEROL-CaCl~ AND CHOL~TERO~M~I~ COMPLEXES WITH ACETIC ANHYDRIDE IN PYRIDINE Samples (300 mg) of the three compounds were suspended in IO ml pyridine and treated with z ml of acetic anhydride. Samples of the reaction mixtures were withdrawn after 0.5, z, 6 and 20 h, diluted with water and extracted with ether. Washed ether phases were used for thin-layer chromatographic analysis of the reaction products. Reference compounds were run simultaneously on the same plates. Average RF values of two experiments were obtained with chloroform as a solvent using Baker-flex Silica Gel rB-F sheets. The spots were classified as strong (s). medium (m) and weak (w) depending on their size and intensity. A medium spot contained approx. qo-60% and a w*sak spot I~_ZO%of the material contained in a strong spot. Sample CaC12 . 2Ha0 complex

RF of thin layer spots after 6h 0.5 h zh

Identification 20

h

0.32 (m)

-

-

0.91 (m) MgCIZ . 6HZ0 complex 0.37 (s)

0.87 (s) -

0.88 Is) -

0.91 (s) -

0.91 (m) 0.40 (s) 0.92 (m)

0.85 (s) 0.32 (s) 0.88 (s)

0.88 (s) 0.30 (w) 0.88 (s)

0.92 (s) 0.86 (s)

Cholesterol

0.35 (s)

cholesterol cholesteryi acetate cholesterol cholesteryl acetate cholesterol cholesteryl acetate

spot of unchang~ cholesterol in case of the MgC12 complex, but in case of the CaCI, complex this spot was weak, representing about 10-20 % of the material in the strong spot. Thus the rate of disappearance of cholesterol was in the following order: cholesterol control > CaClz complex > MgCI, complex. It was not possible to estimate by thin-layer chromatography the rates of formation of the two isomer+ cholesterol dibromides from the two complexes because the sizes and intensities of their spots on thin layers (Com~unds II and III, Table II) were essentially the same, However, the appearance of Compound I, which is an oxidation product of cholesterol dibromide, was in the same order as for the disappearance of cholesterol. The rates of acetylation of cholesterol in the two complexes and in the control sample were found to be in the opposite order to that observed for bromination in ether, namely, MgCl, complex > CaCl, complex > cholesterol. This order is indicated by the thin-layer analysis of samples withdrawn from the reaction mixture after 0.5, 2, 6 and 20 h of reaction (Table III). After 2 h no unreacted cholesterol could be detected in the reaction mixture of the MgClz complex, whereas a medium spot of cholesterol (RF 0.32) was detected in the reaction mixture of the CaCI, complex. The control sample was acetylated relatively slowly. A weak spot of unreacted material was detected even after 6 h of acetylation. Infrared spectra of the two complexes and of cholesterol in the 400-1300 cm-’ region are shown in Fig. I. A comparison of the three spectra reveals slight differences between them with respect to band position, intensity or shape. For example, the position of a weak band is different for the three compounds: rzgo cm-’ (CaCI, complex), 1284 cm-l (MgCl, complex), 1275 Cm-’ (cholesterol). The results obtained on the two complexes and cholesterol control using X-ray powder diffraction are given in Fig. 2. The values of the lattice parameters for the three samples were calculated in each case from 20 low-index diffraction lines and were found to be similar. This result indicated that no major structural rearrangement occurred in the crystals when cholesterol was complexed with either CaCl, or MgCl,. strong

01.

’

1200

’ lxx3 FREQUENCY

I 600 (CM-’

I 600

I

I 400

)

-

28 (DEGREES

1

Fig. I. Infrared spectra of cholesterol-CaC12 . 2H20 (A), cholesterol-MgC12 . 6H20 (B) complexes and of cholesterol (C) obtained on KBr pellets with a Perkin-Elmer Infrared Spectrometer Model 337 with air as the reference. Fig. 2. Diffractometer traces of cholesterol-CaC12 complex (B) and of cholesterol (C).

. 2H,O complex (A), cholesterol-MgCl,

. 6Hz0

However, when the intensities of the diffractometer traces (Fig. 2) were compared, it was found that there were significant differences in the pattern of intensities for the three samples to suggest that there were minor structural differences between them. For example, a diffraction line corresponding to 16.7 A interplanar distance and to 020 Miller index had the following relative intensities: CaCl, complex, 13 ; MgCl, complex, 44; cholesterol control, 65. DISCUSSION

Complex formation between cholesterol and CaCl, . 2H20 or MgC12 * 6H2O occurs in relatively non-polar medium such as 8 % ethanol in acetone. In each case the complex is composed of two molecules of cholesterol and one metal ion. In a biphasic solvent system composed of ether and water the complexes dissociate into cholesterol and the respective salts in less than 5 min after exposure to the solvent. The liberated cholesterol is solubilized in ether and the inorganic salts in water phase. In the absence

of water the complexes are stable and display different solubilities in several organic solvents (Table I). They are, however, much smaller than those of cholesterol control. The solubility of C&l, complex in chloroform is 3.7 mg per ml whereas the MgCl, complex is insoluble in this solvent. While this difference in solubility behavior is interesting and might indicate stronger interaction between cholesterol and Mg2+ and a weaker interaction between cholesterol and Ca2 + in the respective complexes, a more direct approach was necessary to show whether the environment of cholesterol molecule in the two complexes is the same or different. Bromination reaction was chosen as a suitable probe for the study of the steric availability and of chemical reactivity of the A5 doublebond in cholesterol. When a cholesterol control and the two complexes were brominated in ether under identical conditions it was found that the rate of disappearance of cholesterol was greatest for the cholesterol control, followed by CaCl, complex and smallest for the MgCl, complex. This rate was paralleled by the rate of appearance of cholesterol dibromides (5a,6/& and 5/3,6c+dibromides) and the rate of appearance of 5a,6jUibromocholestane -3-one in these reaction mixtures (Table II). Bromination of A5 double bond of cholesterol requires an attack of positively charged Br+ in a direction perpendicular to the plane of the double bond. The vicinity of a positively charged metal ion coordinated with the 3 #Lhydroxyl group of cholesterol and the immediate environment of the A5 double bond in the crystal may influence the rates of bromination. It would appear that in MgC12 complex the inhibitory influence on bromination is greater than in CaCl, complex. Acetylation of the complexes with acetic anhydride in pyridine requires liberation of the 3 /I-hydroxyl group from the coordination sphere of the metal ions if it is to be acetylated by the reagent. When samples of the complexes and of cholesterol were acetylated in pyridine at room temperature, it was found that the rate of acetylation was greatest for MgC12 complex, intermediate for CaCl, complex and slowest for cholesterol (Table III). The rate of disappearance of cholesterol was paralleled by the rate of appearance of cholesteryl acetate. The reason for a greater reactivity of MgC12complex as compared with the other two compounds might be due to an interaction of Mg” with one or both carbonyl oxygens of acetic anhydride molecule, bringing it close to the 3jShydroxyl group of cholesterol and thus facilitating the acetylation. A similar behavior of Mg2+ is encountered in Grignard reactions of ketones [13] and in Gomberg-Bachmann pinacol synthesis [14] in which Mg2+ is believed to coordinate to carbonyl oxygen. This may also imply that the structural relationship between Mg2 ’ and 3j?-hydroxyl group of cholesterol is different in this complex than the relationship between Ca2+ and the same group in the CaCl, complex. The results of spectroscopic measurements performed on the two complexes are in agreement with the results of chemical tests. Thainfrared spectra of the complexes and of cholesterol control (Fig. I) are slightly different from one another in the spectral region 4oo-1300 cm-‘. For example, two strong broad bands located at 550 and at 655 cm-’ are present in the CaCl, complex but are absent in the MgCl, complex. The reverse situation is observed for the broad band located at 625 cm-’ The intensity patterns of Fig. 2 demonstrate the structural changes of the metal complexes (A and B) as compared to cholesterol (C). It can clearly be seen that there is a distinct change in crystallinity when the metal complexes are formed, and

that there are distinct differences between the diffraction patterns of the MgClz and CaCl, complexes. This indicates that Mg*+ and Ca*+ do not assume the same structural relationships with respect to the cholesterol molecule, such as in a typical isomorphic replacement, but rather, that the two ions are in a different environment. The absence in the complexes of the relatively intense high-angle line present in cholesterol is structurally significant. The ability of cholesterol to form molecular complexes is dependent on the availability of its hydroxyl group [3]. When the hydroxyl group is acylated or oxidized to the corresponding ketone, the ability to form complexes is lost. Likewise, the orientation of the hydroxyl group is important. Epicholesterol which, like cholesterol, has themhydroxyl group in the same position but in the opposite orientation (a instead of B) is not able to form complexes. Furthermore, it has been reported [3] that ergosterol, a plant sterol, is also able to form complexes with metallic salts. The structure of ergosterol differs considerably from that of cholesterol. The common structural feature of both compounds, however, is the A ring with the hydroxyl group at C, in j3 configuration. From these facts one may conclude that cholesterol complex formation with metallic salts, including CaCl, and MgCl,, occurs in the vicinity of the hydroxyl group rather than in some other part of the molecule. ACKNOWLEDGEMENTS

The authors express their thanks to Miss Orysia Stanecky for her help with some of the chemical experiments. This research was supported by U.S. Public Health Service Grant No. MH 18165. REFERENCES I Zwikker, J. J. L. (1917) Pharm. Weekbl. 54, 101-102 Hackmann, J. T. (1950) Rec. Trav. Chim. 69, 433-438 3 Garlinskaya, E. I. (1955) J. Appl. Chem. USSR 28, 75-81 4 Parpart, A. K. and Dzieman, A. J. (1940) Cold Spring Harbor Symp. Quant. Biol. 8, 17-24 5 Zlatkis, A., Zak, B. and Boyle, A. J. (1953) J. Lab. Clin. Med. 41, 486-492 6 Windaus, A. (1906) Berichte 39, 518-523 7 Barton, D. H. R. and Miller, E. (1950) J. Am. Chem. Sot. 72, 1066-1070 8 Fieser, L. F. (1953) J. Am. Chem. Sot. 75, 5421-5422 9 Weast, R. C. (1968) Handbook of Chemistry and Physics, p. C251, The Chemical Rubber Company, Cleveland IO Azaroff, L. V. and Buerger, M. J. (19.58) The Powder Method in X-ray Crystallography, pp. 176179, McGraw-Hill Book Co. Inc., New York II Edsall, J. T. and Wyman, J. (1958) Biophysical Chemistry, Vol. I, p. 44, Academic Press, New York 12 Rauen, H. M. (1964) Biochemisches Taschenbuch, Part 2, p. I IO, Springer-Verlag, Berlin 13 Miller, J., Gregoriou, G. and Mosher, H. S. (1961) J. Am. Chem. Sot. 83, 3966-3971 14 March, J. (1968) Advanced Organic Chemistry: Reactions, Mechanisms and Structure, p. 903, McGraw-Hill Book Co. Inc., New York 15 Weichherz, J. and Marschik, H. (1932) Biochem. Z. 249, 312-322 2