PROPERTIES OF CHOLESTEROL OBTAINED FROM DIFFERENT SOURCES.

BY R. J. ANDERSON.

(From the Biochemical Laboratory, New York Agricultural Experiment Station, Geneva.)

(Received for publication, November 12, 1926.)

INTRODUCTION.

The question of the origin of cholesterol has not been settled. Unicellular organisms are apparently capable of synthesizing sterols as well as other cell constituents from simpler organic compounds. In the case of animals it is believed that they uti- lize plant sterols (1) and that in some manner somewhere in the body phytosterol is changed to the isomeric compound, cholesterol, which only varies from the former in physical properties. Experi- mental evidence has also been presented (2) which indicates that cholesterol may be synthesized in the animal body.

It would seem, however, from recent work on plant sterols (3) that more far reaching molecular rearrangements must be neces- sary in order that phytosterols may be changed into cholesterol than has been supposed heretofore. To consider only one plant fat, for instance that from corn germ (4), it has been found that the crystalline sterol isolated from it is a complex mixture con- sisting of the saturated dihydrositosterol, the unsaturated stig- master01 with two double bonds, and at least three isomeric sitosterols with one double bond each. That these different phytosterols, which vary not only in physical properties but also in composition, could all be changed into one single cholesterol does not seem likely. It may be necessary therefore to reckon with the possibility of a number of isomeric cholesterols in animal fats.

In connection with the investigation conducted by Bloor (5) on the fatty acids of the plasma of beef and dog blood, considerable

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408 Properties of Cholesterol

quantities of unsaponifiable material were obtained. These con- stituents were kindly given us by Dr. Bloor for examination.

A decided difference in the physical appearance of the crude material obtained from beef and dog plasma was noticed, and even the purified cholesterols showed slight differences in melting points and in optical rotation. The substance from beef plasma consisted almost entirely of crystalline cholesterol which after it had been purified appeared to be homogeneous. Although the animals had received only plant sterols in their food, there was no evidence of the presence of phytosterols in the blood plasma. The transformation of phytosterols into cholesterol, if it occurs, must therefore take place very quickly.

The unsaponifiable matter from the plasma of dog blood con- tained some 40 per cent of non-crystalline semisolid material that could not be eliminated from the cholesterol by crystallization from alcohol. Precipitation with digitonin did not lead to a pure cholesterol because a part of the non-crystalline substance was also precipitated. Pure cholesterol could only be obtained by means of the dibromo- compound.

The fact that a cholesterol preparation appears to be homogene- ous, i.e. recrystallization causes no change in the properties, is no absolute proof of the purity. Closely allied sterols may differ so slightly in solubility that recrystallization causes no perceptible separation. It has been shown that a mixture of isomeric sito- sterols (6) may be recrystallized at least ten times without show- ing any notable change in properties.

In order to determine whether different cholesterol preparations are homogeneous it will be necessary to prepare cholesterol esters of various acids or some other derivatives of cholesterol and frac- tionally recrystallize these compounds, and to compare the proper- ties of the different fractions. As a beginning in this direction we have subjected cholesterol acetate to fractional crystallization from ethyl alcohol. The results indicate that commercial cho- lesterol contains a small amount of some substance which causes a decided lowering of the melting point and of the optical rotation of the bottom fraction. More experimental work will be necessary before one can determine whether the depression of the melting point and optical rotation is due to the presence of an accidental im- purity or if it is due to some compound isomeric with cholesterol.

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R. J. Anderson 409

EXPERIMENTAL PART.

Cholesterol from Beef Plasma.

The crude unsaponifiable matter was supplied by Dr. W. R. Bloor and was obtained as a by product from his work on the Fatty Acids of Blood Plasma (5). It had been prepared as fol- lows: The plasma was digested in about 20 per cent sodium hy- droxide at 90°C. for 8 hours, after which the mixture was cooled, acidified with hydrochloric acid, and the lipid material was ex- tracted with ether. The fatty acids were separated from the unsaponifiable matter by first dissolving the mixture in petroleum ether and then adding successively equal volumes of alcoholic potassium hydroxide and of water. The fatty acids dissolved in

TABLE I.

Proper&s of Different Fractions of Cholesterol from Beef Plasma.

Fract,ion No. Weight. M. p.’ Solidifying point. k&i

OWL “C. “C.

I 15.5 149 118 -39.52” 2 6.2 149 118 -39.40” 3 1.1 149 116 -39.71”

* All melting points given in this paper ape corrected. 1 All optical rotations were determined in chloroform solution and with

sodium light.

the alkaline alcohol and water while the unsaponifiable substance remained in the petroleum ether. After a second extraction with petroleum ether the combined extracts were washed with water, the solvent distilled off, and the residue was dried.

The crude cholesterol, which weighed 25 gm., was yellow in color and consisted of large irregular plate-shaped crystals. The material was dissolved in 700 cc. of hot alcohol, boiled with norit, and filtered. As the solution cooled the cholesterol separated in very large, thin, colorless plates that filtered very slowly on a Buchner funnel. The substance was recrystallized three times, using about 600 cc. of alcohol for each crystallization. When the solution was cooled rapidly t.he cholesterol crystallized in small uniform plates. After drying at 60°C. the crystals weighed 15.5

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410 Properties of Cholesterol

gm. This is designated Fraction 1 in Table I. Fraction 2 was obtained by concentrating the above mentioned mother liquor and recrystallizing the substance four times. Fraction 3 was obtained on concentrating the last mother liquors and recrystal- lizing the product nine times until a colorless preparation resulted. The properties of these fractions are shown in Table I.

Fraction 1 was analyzed after it had been dried at 105°C. in a vacuum over phosphorus pentoxide.

Analysis. 0.1505 gm. substance: 0.1604 gm. HSO and 0.4622 gm. COZ.

Calculated for C&,H,,OH (386). C 83.93, H 11.91 per cent. Found. C 83.75, H 11.92 per cent.

The properties of the three fractions described in Table I were practically identical and the composition agreed with the usual formula for cholesterol.

TABLE II.

Optical Rotation of Cholesterol and Cholesteryl Acetate in Chloroform and Ether.

Cholesterol. Cholesteryl acetate.

[& in CHCls. [aID in ether. [orID in CHCL. [aID in ether.

-39.52” -31.46” --3’1.60” -43.11” -30.90” -30.54”

Acetyl Derivative.

The acetyl derivative was prepared by boiling 4 gm. of Fraction 1 with 30 cc. of acetic anhydride for 1.5 hours. After cooling, the crystals were filtered off, washed with cold methyl alcohol, and recrystallized three times from ethyl alcohol. The substance separated in fine colorless needles which after drying in the air weighed 3.7 gm. It melted at 116” and on cooling a fine display of colors was observed. The rotation was -43.11”.

Variation in Optical Rotation in Di$erent Xolvents.

The optical rotation of cholesterol and of cholesteryl acetate varies greatly with the solvent. A comparison of the optical activity in chloroform and ether is given in Table II.

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R. J. Anderson 411

The levorotation is depressed in ethereal solution and the intro- duction of the negative acetyl group which causes an increase in levorotation in chloroform gives a lower value in ether than does free cholesterol.

Reduction of Cholesterol.

p-Cholestanol has been prepared and studied by several investi- gators, having first been described by Diels and Abderhalden (7). It was first shown by WillstSltter and Meyer (8) that the same substance was formed when cholesterol was reduced by hydrogen in the presence of platinum black. /3-Cholestanol has since that time been prepared by a number of investigators (9) and the melting point is usually given as 141-142’ while the re- corded rotations vary from +23’ to f34”.

The cholesterol, 5 gm. of Fraction 1, was reduced with hydrogen and platinum oxide as described for sitosterol (10). The P-cho- lestanol was recrystallized several times from alcohol from which it separated in large colorless plates containing 1 molecule of water of crystallization. In the Liebermann-Burchard reaction no immediate coloration was given but after the reaction mixture had stood for some time a faint bluish color developed. The substance melted at 143’. The rotation was determined in chloroform and in ether with the following results:

In chloroform solution, [C-Z]=, +23.56’, f23.45”. In ethereal solution, [LY]~, +27.07”, +27.06”.

Acetyl Derivative of fi-Cholestanol.

The acetyl derivative was prepared and twice recrystallized from alcohol. It melted at 111’ and the rotation in chloroform solution was +13.27”.

Examination of UnsaponiJiable Matter from Normal Dog Blood Plasma.

The crude material was supplied by Dr. W. R. Bloor and it had been obtained in a similar way to that from beef blood plasma. The substance, which weighed 16.3 gm., was a yellow crystalline solid that obviously contained much oily material. It possessed a

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412 Properties of Cholesterol

slight but peculiarly unpleasant odor. The substance was dis- solved in 400 cc. of hot alcohol and treated twiqe with norit, which did not remove much of the coloring matter. After six recrystal- lizations the top fraction was snow-white, but the crystals were not homogeneous; two forms of crystals were evident, viz. colorless plates that somewhat resembled cholesterol and also branching fern-like crystals. The substance weighed 1.9 gm., m.p., 143- 144”.

The yellow colored mother liquors from the above mentioned crystallizations now contained most of the original material in solution. It was again treated with norit, filtered, and concen- trated to about 600 cc. On standing at room temperature 4 gm. of colorless plate-shaped crystals separated, m.p., 151-152’. The filtrate was concentrated to about 200 cc. and allowed to cool when 6 gm. of slightly yellow crystals were collected, m.p., 149-150’.

The last mother liquor deposited, on standing for several days, about 50 mg. of yellow octahedral crystals that were identified as sulfur. After the above mentioned crystals had been filtered off, the filtrate was evaporated to dryness when a yellow wax-like material of unpleasant odor remained.

The appearance of the various crystalline fractions, obtained as described above, together with the variations in melting point indicated that the material was very impure and it seemed to be impossible to obtain any pure cholesterol by crystallization. ,4n attempt was therefore made to precipitate the cholesterol from the mixture by means of digitonin according to the method of Windaus (11). Portions of 4.5 gm. of the impure cholesterol were dissolved in 400 cc. of hot alcohol and mixed with a solution containing 15 gm. of digitonin in 1500 cc. of boiling alcohol. Fine colorless flakes or plate-shaped crystals began to separate at once and the mixture, after cooling, was allowed to stand in the ice box overnight. The crystalline precipitate was filtered off, washed thoroughly with alcohol and ether, and dried in a vacuum desiccator over sulfuric acid. The dry digitonide was extracted with boiling xylene as described by Windaus (12). The digitonin recovered in this manner was used over again until all of the crude cholesterol had been precipitated.

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R. J. Anderson 413

Examination of Material Not Precipitated by Digitonin.

The alcoholic mother liquors from the digitonide were evaporated to dryness under reduced pressure. The residue was extracted with ether; the ethereal solution was filtered and the ether was distilled. The residue was a yellow oil of unpleasant odor which on standing at room temperature gradually deposited a few octa- hedral crystals; a part separated as long thin flakes and the whole formed a semisolid mass that weighed 3 gm. It is evident there- fore that about 18 per cent of the original unsaponifiable matter was not precipitated by digitonin. The material was treated with 60 cc. of hot alcohol which dissolved everything except the yellow octahedral crystals. The latter were identified as sulfur. The clear alcoholic solution was allowed to cool when a part of the dissolved substance separated as an oily layer on the bottom of the flask. The solution was decanted and evaporated to dryness when a yellow oil was obtained. It was impossible to secure any crystals from either of the above mentioned fractions. Both substances gave the Liebermann-Burchard reaction in the same manner as pure cholesterol and in chloroform solution bromine was absorbed. It is probable therefore that the material was closely related to cholesterol although it was not precipitated by digitonin.

The substance was reduced with hydrogen and platinum oxide as described for cholesterol. A considerable amount of hydrogen was absorbed but the reduction product, on evaporation of the solvent, formed a yellow oil that solidified on standing to a thick gummy mass that could not be crystallized from any of the usual organic solvents. In the Liebermann-Burchard reaction it gave a slight greenish yellow fluorescent coloration.

Examination of the Cholesterol that Had Been Precipitated with Digitonin.

The cholesterol which remained in the xylene, after decomposing the digitonide, was obtained by distilling the solvent under re- duced pressure and after the last traces of xylene had been removed by distillation with steam. After the solution had cooled the cholesterol was filtered off, washed with water, and dried in a vacuum desiccator over sulfuric acid. The material was a slightly sticky solid that weighed 13.3 gm. The substance was therefore

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414 Properties of Cholesterol

recovered quantitatively. It was dissolved in 180 cc. of hot aIco- hol, treated with norit, and recrystallized from alcohol when 5.1 gm. of ball-shaped aggregates of thick plates were obtained that did not resemble cholesterol crystals. The substance sintered af 146’ and melted at 149”. A second fraction of impure cholesterol weighing 4.1 gm. was obtained by concentrating the mother liquors.

After the last mentioned crystals had been filtered off, the filtrate was evaporated to dryness. The residue which weighed 3 gm. was a yellow wax-like material. This residue which showed no< tendency to crystallize evidently did not contain very much cholesterol because when brominated by the method of Windaus and Hauth (13) no crystals of cholesterol dibromide were obtained. The original unsaponifiable matter contained therefore 6 gm. or about 37 per cent of non-crystalline material, the nature of which is unknown.

PuriJication of Cholesterol by means of the Dibromide.

The results described above indicate that the unsaponifiable. matter obtained from the plasma of normal dog blood contains. several substances in addition to cholesterol. A part of this. material did not react with digitonin while a portion of non- crystalline substance was precipitated with this reagent along with. cholest,erol.

Since it appeared to be impossible to secure any pure cholesterol. by crystallization, the two fractions of impure crystals described above were united and brominated by the method of Windaus and Hauth (13). The bromination mixture contained an excess of. bromine but no hydrobromic acid could be detected. The cho- lesterol dibromide began to crystallize immediately in colorless. needles. After the solution had stood in a freezing mixture for some time the crystals were filtered off, washed with glacial acetic- acid, and finally with water. The snow-white substance, after- it had been dried in a vacuum desiccator over sulfuric acid, weighed 8.4 gm. When heated in a capillary tube, it softened at llS” and melted to a brown liquid at 122’. The rotation in chloro- form solution was -43.53”.

The substance was debrominated by boiling its alcoholic solu-

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R. J. Anderson 415

tion with zinc dust and acetic acid and the reaction product was recrystallized five times from alcohol. The snow-white plates had the characteristic form of cholesterol crystals and after drying in the air weighed 3.2 gm. The substance melted at 150-151’ and the melt solidified at 119’; [a],, -38.11’. From the mother liquors a second fraction of cholesterol was obtained which melted at 150-151’; [aJD, -38.22’. This cholesterol preparation had a slightly higher melting point but the rotation was about lo lower than the cholesterol isolated from beef blood plasma.

The acetyl derivative was prepared and it was twice recrystal- lized from alcohol. It separated in burr-shaped aggregates of colorless needles, m.p. 115’; [G]~, -42.56’.

Examination of Commercial Cholesterol.

Cholest,erol’ of unknown origin was used in these experiments. An alcoholic solution containing 100 gm. of cholesterol and 30 gm. of potassium hydroxide was refluxed for 2 hours. One half of the .alcohol was distilled off and the cholesterol was precipitated by adding water. The mixture was extracted with ether and the ethereal solution, after washing four times with water, was dried with sodium sulfate and filtered. The filtrate, which was yellowish in color and not quite clear, was shaken with norit and filtered when a colorless solution was obtained. The ether was distilled leaving a snow-white residue which was recrystallized from 700 cc. of alcohol when colorless plates characteristic of cholesterol crystals were obtained. The mother liquor was concentrated and the material that separated was recrystallized from alcohol, giv- ing colorless plates that appeared to be identical with the first lot. The properties of the two fractions were practically identical as shown in Table III.

Acetyl Derivative.

The acetyl derivative was prepared by boiling 50 gm. of the purified cholesterol with 200 cc. of acetic anhydride for 1 hour. After cooling, the crystals were filtered off and washed with cold methyl alcohol. The snow-white product was recrystallized from

1 Purchased from Eastman Kodak Company.

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416 Properties of Cholesterol

alcohol when small colorless needles were obtained. The sub- stance melted at 116-117”, solidified at 108”; [a],, -43.65’.

This preparation was recrystallized ten times and separated into four fractions. The first two mother liquors were concentrated, yielding Fraction 4; the next three mother liquors gave Fraction 3, while Fraction 2 was obtained by concentrating the last five mother liquors. The properties of these fractions are shown in Table IV.

The top fraction was identical in properties with the original acetyl derivative but the other fractions showed slightly lower rotations and melting points.

TABLE III.

Properties of Purijied Cholesterol.

M. p. Solidifying pcint. [aID in CHCh. Water of crystallization.

“C. “C. per cent 150-151 120 -39.72” 5.33 15cb151 116 -39.63” 5.24

TABLE IV.

Fractionation of Cholesteryl Acetate.

Fraction No. Weight. M.P. [aID in CHCL.

~~-

wn. “C.

1 32.7 116 -43.59” 2 10.0 115-116 -43.39” 3 4.4 115-116 -42 92” 4 5.0 114116 -42 62’

Fraction 4 which possessed the lowest melting point and rotation was again twice recrystallized from alcohol and separated into two fractions. The bottom fraction was obtained by concentrat- ing the mother liquors. It crystallized in colorless needles and melted at 111-112’. The rotation in chloroform solution was -41.12”.

The results indicate a marked difference in the properties. between the top fraction in Table IV and the last bottom fraction. The variation in melting point and rotation must be due to some- substance having a lower rotation t.han ordinary cholesterol. It is evident, however, that only a small amount of this substance

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R. J. Anderson

could have been present. In order to determine the nature of the contaminating substance it will be necessary to fractionate larger quantities of cholesterol. It is not improbable that some deriva- tive other t,han the acetate and that a solvent better than alcohol could be found that would cause a more effective separation, but lack of time has prevented further investigation along these lines.

In conclusion the author takes pleasure in acknowledging his indebtedness to Dr. W. R. Bloor of the University of Rochester, for generously supplying crude cholesterol.

SUMMARY.

Attention is called to the fact that cholesterol preparations obtained from different sources show slight differences in physical properties.

When apparently pure cholesteryl acetate is fractiona,lly recrys- tallized from ethyl alcohol, it is possible t,o separate a bottom frac- tion that possesses a much lower melting point and a lower optical rotation than the top fraction.

If cholesterol is formed from plant sterols a number of different as well as isomeric cholesterols might be expected to occur in animal fats corresponding to the various phytosterols contained in the plant material which serves as food.

BIBLIOGRAPHY.

1. Fraser, M. T., and Gardner, J. A., Proc. Roy. Sot. London, Series B, 1909, Ixxxi, 230; 1909-10, lxxxii, 559.

2. Gardner, J. A., and Fox, F. W., Proc. Roy. Sot. London, Series B, 1921, xcii, 358. Beumer, H., and Lehmann, F., Z. ges. exp. Med., 1923, xxxvii, 274. Channon, H. J., Biochem. J., 1925, xix, 424. Randles, F. S., and Knudson, A., J. Biol. Chem., 1925, lxvi, 459.

3. ‘Anderson, R. J., and Shriner, R. L., J. Am. Chem. Sot., 1926, xlviii, 2976. Nabenhauer, F. P., and Anderson, R. J., J. Am. Chem. Sot., 1926, xlviii, 2972. Anderson, R. J., Shriner, R. L., and Burr, G. O., J. Am. Chem. Sot., 1926, xlviii, 2987.

4. Anderson, R. J., and Shriner, R. L., J. Am. Chem. Sot., 1926, xlviii, 2976. 5. Bloor, W. R., J. Biol. Chem., 1923, lvi, 711; 1924, lix, 543. 6. Anderson, R. J., and Shriner, R. L., J. Am. them. Sot., 1926, xlviii, 2976. 7. Diels, O., and Abderhalden, E., Ber. them. Ges., 1906, xxxix, 884. 8. Willstatter, R., and Meyer, E. W., Ber. them. Ges., 1908, xii, 2199.

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9. DorCe, C., J. Chem. SOL, 1909, 638. Boehm, R., Biochem. Z., 1911, xcv, xxxiii, 474. Windaus, A., and Uibrig, C., Ber. them. Ges., 1914, xlvii, 2384. von Fiirth, O., and Felsenreich, G., Biochem. Z., 1915, lxix, 420. Ellis, G. W., and Gardner, J. A., Biochem. J., 1918, xii, 72. Nord, F. F., Biochem. Z., 1919, xcix, 265.

10. Anderson, R. J., and Nabenhauer, F. P., J. Am. Chem. Xoc., 1924, xlvi,. 1953.

11. Windaus, A., Ber. them. Ges., 1909, xlii, 238; 2. physiol. Chem., 1910, lxv, 110.

12. Windaus, A., 2. physiol. Chem., 1910, lxv, 110. 13. Windaus, A., and Hauth, A., Ber. them. Ges., 1906, xxxix, 4378.

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R. J. AndersonSOURCES

OBTAINED FROM DIFFERENT PROPERTIES OF CHOLESTEROL

1927, 71:407-418.J. Biol. Chem. 

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