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PROPERTIES OF CHOLESTEROL OBTAINED FROMDIFFERENT SOURCES.

BY R. J. ANDERSON.(From the Bioc hem ical 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 synthesizingsterols as well as other cell constituents from simpler organiccompounds. In the case of animals it is believed that they uti-lize plant sterols (1) and that in some manner somewhere in thebody 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 thatcholesterol 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 cholesterolthan has been supposed heretofore. To consider only one plantfat, for instance that from corn germ (4), it has been found thatthe 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 isomericsitosterols with one double bond each. That these differentphytosterols, which vary not only in physical properties but alsoin composition, could all be changed into one single cholesteroldoes not seem likely. It may be necessary therefore to reckonwith the possibility of a number of isomeric cholesterols in animalfats.

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 Cholesterolquantities 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 crudematerial obtained from beef and dog plasma was noticed, andeven the purified cholesterols showed slight differences in meltingpoints and in optical rotation. The substance from beef plasmaconsisted almost entirely of crystalline cholesterol which after ithad been purified appeared to be homogeneous. Although theanimals had received only plant sterols in their food, there was noevidence of the presence of phytosterols in the blood plasma. Thetransformation of phytosterols into cholesterol, if it occurs, musttherefore take place very quickly.

The unsaponifiable matter from the plasma of dog blood con-tained some 40 per cent of non-crystalline semisolid material thatcould not be eliminated from the cholesterol by crystallizationfrom alcohol. Precipitation with digitonin did not lead to a purecholesterol because a part of the non-crystalline substance wasalso precipitated. Pure cholesterol could only be obtained bymeans 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 noabsolute proof of the purity. Closely all ied sterols may differ soslightly in solubility that recrystallization causes no perceptibleseparation. 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 preparationsare homogeneous it will be necessary to prepare cholesterol estersof 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 wehave subjected cholesterol acetate to fractional crystallizationfrom ethyl alcohol. The results indicate that commercial cho-lesterol contains a small amount of some substance which causes adecided lowering of the melting point and of the optical rotation ofthe bottom fraction. More experimental work will be necessarybefore one can determine whether the depression of the meltingpoint 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 409EXPERIMENTAL 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 90C. for 8 hours, after which the mixture was cooled,acidified with hydrochloric acid, and the lip id material was ex-tracted with ether. The fatty acids were separated from theunsaponifiable matter by first dissolving the mixture in petroleumether and then adding successively equal volumes of alcoholicpotassium hydroxide and of water. The fatty acids dissolved in

TABLE I.Proper&s of Different Fractions of Cholesterol from Beef Plasm a.

Fract,ion No. Weight. M. p. Solidifyingpoint. k& iOWL C. C.

I 15.5 149 118 -39.522 6.2 149 118 -39.403 1.1 149 116 -39.71

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

sodium light.the alkaline alcohol and water while the unsaponifiable substanceremained in the petroleum ether. After a second extraction withpetroleum 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 incolor and consisted of large irregular plate-shaped crystals. Thematerial was dissolved in 700 cc. of hot alcohol, boiled with norit,and filtered. As the solution cooled the cholesterol separated invery large, thin, colorless plates that filtered very slowly on aBuchner funnel. The substance was recrystallized three times,using about 600 cc. of alcohol for each crystallization. When thesolution was cooled rapidly t.he cholesterol crystallized in smalluniform plates. After drying at 60C. the crystals weighed 15.5

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410 Properties of Cholesterolgm. This is designated Fraction 1 in Table I. Fraction 2 wasobtained by concentrating the above mentioned mother liquorand recrystallizing the substance four times. Fraction 3 wasobtained 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 105C. ina 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 werepractically identical and the composition agreed with the usualformula for cholesterol.TABLE II.Optical Rotation of Cholesterol and Cholesteryl Acetate in Chloroformand Ether.

Cholesterol. Cholesteryl aceta te.[& in CHCls. [aID in ether. [orID in CHCL. [aID in ether.

-39.52 -31.46 --31.60 -43.11 -30.90 -30.54

Acetyl Derivative.The acetyl derivative was prepared by boiling 4 gm. of Fraction1 with 30 cc. of acetic anhydride for 1.5 hours. After cooling, thecrystals were filtered off, washed with cold methyl alcohol, andrecrystallized three times from ethyl alcohol. The substanceseparated in fine colorless needles which after drying in the airweighed 3.7 gm. It melted at 116 and on cooling a fine displayof colors was observed. The rotation was -43.11.

Variation in Optical Rotation in Di$erent Xolvents.The optical rotation of cholesterol and of cholesteryl acetatevaries greatly with the solvent. A comparison of the opticalactivity in chloroform and ether is given in Table II.

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R. J. Anderson 411The levorotation is depressed in ethereal solution and the intro-

duction of the negative acetyl group which causes an increase inlevorotation in chloroform gives a lower value in ether than doesfree 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 thesame substance was formed when cholesterol was reduced byhydrogen in the presence of platinum black. /3-Cholestanol hassince 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 hydrogenand platinum oxide as described for sitosterol (10). The P-cho-lestanol was recrystallized several times from alcohol from whichit separated in large colorless plates containing 1 molecule of waterof crystallization. In the Liebermann-Burchard reaction noimmediate coloration was given but after the reaction mixture hadstood for some time a faint bluish color developed. The substancemelted at 143. The rotation was determined in chloroform and inether 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 recrystallizedfrom alcohol. It melted at 111 and the rotation in chloroformsolution was +13.27.Examination of UnsaponiJiable Matter from Normal Dog Blood

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

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412 Properties of Cholesterolslight but peculiarly unpleasant odor. The substance was dis-solved in 400 cc. of hot alcohol and treated twiqe with norit, whichdid not remove much of the coloring matter. After six recrystal-lizations the top fraction was snow-white, but the crystals were nothomogeneous; two forms of crystals were evident, viz. colorlessplates that somewhat resembled cholesterol and also branchingfern-like crystals. The substance weighed 1.9 gm., m.p., 143-144.

The yellow colored mother liquors from the above mentionedcrystallizations now contained most of the original material insolution. It was again treated with norit, filtered, and concen-trated to about 600 cc. On standing at room temperature 4 gm. ofcolorless plate-shaped crystals separated, m.p., 151-152. Thefiltrate was concentrated to about 200 cc. and allowed to cool when6 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 assulfur. After the above mentioned crystals had been filtered off,the filtrate was evaporated to dryness when a yellow wax-likematerial of unpleasant odor remained.

The appearance of the various crystalline fractions, obtained asdescribed above, together with the variations in melting pointindicated that the material was very impure and it seemed to beimpossible to obtain any pure cholesterol by crystallization. ,4nattempt was therefore made to precipitate the cholesterol fromthe mixture by means of digitonin according to the method ofWindaus (11). Portions of 4.5 gm. of the impure cholesterolwere dissolved in 400 cc. of hot alcohol and mixed with a solutioncontaining 15 gm. of digitonin in 1500 cc. of boiling alcohol. Finecolorless flakes or plate-shaped crystals began to separate at onceand the mixture, after cooling, was allowed to stand in the icebox overnight. The crystalline precipitate was filtered off,washed thoroughly with alcohol and ether, and dried in a vacuumdesiccator over sulfuric acid. The dry digitonide was extractedwith boiling xylene as described by Windaus (12). The digitoninrecovered in this manner was used over again until all of the crudecholesterol had been precipitated.

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R. J. Anderson 413Examination of Material Not Precipitated by Digitonin.

The alcoholic mother liquors from the digitonide were evaporatedto dryness under reduced pressure. The residue was extractedwith ether; the ethereal solution was filtered and the ether wasdistilled. The residue was a yellow oil of unpleasant odor whichon standing at room temperature gradually deposited a few octa-hedral crystals; a part separated as ong thin flakes and the wholeformed a semisolid mass hat weighed 3 gm. It is evident there-fore that about 18 per cent of the original unsaponifiable matterwas not precipitated by digitonin. The material was treatedwith 60 cc. of hot alcohol which dissolved everything except theyellow octahedral crystals. The latter were identified as sulfur.The clear alcoholic solution was allowed to cool when a part of thedissolved substance separated as an oily layer on the bottom of theflask. The solution was decanted and evaporated to dryness whena yellow oil was obtained. It was impossible to secure any crystalsfrom either of the above mentioned fractions. Both substancesgave the Liebermann-Burchard reaction in the same manner aspure cholesterol and in chloroform solution bromine was absorbed.It is probable therefore that the material was closely related tocholesterol although it was not precipitated by digitonin.The substance was reduced with hydrogen and platinum oxideas described for cholesterol. A considerable amount of hydrogenwas absorbed but the reduction product, on evaporation of thesolvent, formed a yellow oil that solidified on standing to a thickgummy mass hat could not be crystallized from any of the usualorganic solvents. In the Liebermann-Burchard reaction it gavea slight greenish yellow fluorescent coloration.Examination of the Cholesterol that Had Been Precipitated withDigitonin.

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

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414 Properties of Cholesterolrecovered quantitatively. It was dissolved in 180 cc. of hot aIco-hol, treated with norit, and recrystallized from alcohol when 5.1gm. of ball-shaped aggregates of thick plates were obtained thatdid not resemble cholesterol crystals. The substance sintered af146 and melted at 149. A second fraction of impure cholesterolweighing 4.1 gm. was obtained by concentrating the motherliquors.

After the last mentioned crystals had been filtered off, the filtratewas evaporated to dryness. The residue which weighed 3 gm.was a yellow wax-like material. This residue which showed no