PROTEOLIPIDES, A NEW TYPE OF TISSUE LIPOPROTEINS ·  · 2003-02-25PROTEOLIPIDES, A NEW TYPE OF TISSUE LIPOPROTEINS THEIR ISOLATION FROM BRAIN* BY JORDI FOLCH AND M. LEES? (From the

PROTEOLIPIDES, A NEW TYPE OF TISSUE LIPOPROTEINS

THEIR ISOLATION FROM BRAIN*

BY JORDI FOLCH AND M. LEES?

(From the McLean Hospital Research Laboratories, Waverley, Massachusetts, and the Department of Biological Chemistry, Harvard Medical School,

Boston, Massachusetts)

(Received for publication, February 3, 1951)

Brain tissue and brain tumor tissue have been found to contain a new type of component to which the name of proteolipides has been given (1). Proteolipides are lipoproteins having as constituents a lipide moiety and a protein moiety, but, while other known lipoproteins are soluble in water or salt solutions, proteolipides are insoluble in water and soluble in chloro- form-methanol mixtures; i.e., their solubilities are akin to those of lipides. It is to emphasize this special feature that the name proteolipides has been coined.

Proteolipides are found in all tissues that have been studied, with the exception of blood plasma. The tissue that is richest in them is white matter of brain in which proteolipide protein constitutes 2.0 to 2.5 per cent of the weight of wet tissue. Gray matter is the second richest tissue in them, closely followed by heart muscle. Kidney, liver, lung, skeletal muscle, and smooth muscle contain proteolipides in smaller amounts than do heart muscle or than brain. Brain tumors range between white matter and gray matter in proteolipide content.

Most of the work reported here has been carried out on white matter. From it have been isolated three different proteolipide fractions, one of which was obtained consistently as a crystalline substance.

The first indication of the existence of proteolipides was the observation that lipide extracts prepared from brain tissue, by a method described in an accompanying paper (Z), and presumably free of non-lipide contami- nants, contained more N than could be expected from their phosphatide and cerebroside content. In an attempt to isolate the lipide fraction which contained this unidentified lipide N, it was found that, on drying

* This work has been aided by a grant from the National Institutes of Health, United States Public Health Service, and grants from the Bmerican Cancer Society. Part of the data in this paper is taken from a thesis to be submitted by M. Lees to the Division of Medical Sciences of Harvard University in partial fulfilment for the degree of Doctor of Philosophy.

t Predoctoral Fellow of the National Institutes of Health, United States Public Health Service, 1947-50.

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the extract by vacuum distillation of the solvents, part of the residue had become insoluble in the same chloroform-methanol mixture, 2: 1 by volume, used for the original extraction of the tissue. The part of the residue that had become insoluble in this solvent mixture as a result of drying was also insoluble in water. It was found to be protein material.

Further study established (a) that the amount of protein extracted with chloroform-methanol under the conditions used was always the same from the same tissue and that it remained constant in spite of freezing and thawing the tissue prior to extraction or carrying out the extraction at different temperatures within the range of -15” to the boiling point of the solvent mixture (about 59’); (b) that taking the extract to dryness, even under the mildest possible conditions, resulted invariably in render- ing all of the protein present in the extract insoluble in the chloroform- methanol mixture; and (c) that washing the extract with water to free it of non-lipide contaminants, as described elsewhere (2), did not result in any removal of protein from the extract.

In view of what is known about the properties of proteins, the foregoing facts made it necessary to assume that the protein material being dealt with was not a free protein but that it was combined with lipides in such a way that the resulting compound or compounds offered an outside lipide surface. A study of possible procedures of isolation was started. Taking the extract to dryness was out of the question, since it invariably resulted in rendering the protein present insoluble in chloroform-methanol mix- tures. Attempts at fractionation by addition of different solvents to the extract proved unsuccessful. They resulted either in separating the pro- tein in a form insoluble in chloroform-methanol mixtures or in the separa- tion of fractions of protein content similar to that of the extract itself. The following procedure was finally developed: A chloroform-methanol extract is placed in contact with at least 5-fold its volume of water. The material (‘Yluff”) which collects at the interphase is freely soluble in chlo- roform-methanol. From it two fractions’ (proteolipides A and B) have been separated. The underlying chloroform solution can be concentrated to dryness without the protein losing its solubility. From the residue, a third proteolipide (C) has been separated.

Details of the procedure of isolation of proteolipides A, B, and C and some information gathered on their properties and chemical composition are given in the experimental part. Proteolipide A is a mixture. Pro- teolipide B has been consistently obtained as a crystalline material and may or may not be a pure substance. It contains 50 per cent protein and 50 per cent lipides, the lipides being a mixture in equal proportions of phosphatides and cerebrosides. About half of the phosphatides appear to be sphingomyelin as estimated by the method of Schmidt et al. (3). Pro-

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teolipide C contains 75 per cent protein and 25 per cent lipides. The lipides are mostly phosphatides of the phosphoglyceride type. All three proteolipides are birefringent white powders, insoluble in water and soluble in chloroform-methanol mixtures which contain an amount of water close to the saturation point. Proteolipide C is also soluble in pure chloroform. All three proteolipides can be split into their lipide and protein constituent moieties by dissolving them in chloroform-methanol-water mixtures and removing the solvents by vacuum distillation. On extracting the residue obtained with the same solvent mixture, the lipides dissolve, leaving be- hind a protein residue. In the case of proteolipides A and B, this pro- cedure results in complete separation of the two moieties.; in the case of proteolipide C, between one-fourth and one-third of the protein still re- mains attached to lipides as a chloroform-soluble compound. The mech- anism of action of this “denaturation” appears to depend on the separa- tion of the solvent mixture into two phases during the removal of the solvents by vacuum distillation. It is possible that proteolipides, which have the property of accumulating at interphases, are less stable when at an interphase than when in solution or in the solid state.

The protein moiety from each of the three proteolipides contains 1.76 per cent S and is resistant to tryptic action. Neurokeratin (4), a protein fraction prepared from brain tissue by prolonged extraction with solvents, followed by treatment of the tissue residue with trypsin and pepsin, ex- hibits the same features. Extraction of brain tissue by solvents under conditions different from those described in this paper results in incomplete extraction of proteolipide protein (2). Therefore, it is quite likely that what has been described as neurokeratin is for the most part the protein moiety of proteolipides.

No exact information has been obtained on the nature of the bond be- tween lipide and protein in proteolipides. It does not depend for its in- tegrity on the presence of bound water (there is no loss in weight of pro- teolipides when they are heated at 100’ in vacua). The fact that all proteolipides are birefringent suggests that the spatial relationship between the constituent moieties is all important.

EXPERIMENTAL

Analytical Methods-Most of the analytical methods used have been described elsewhere (5). P has been estimated by the method of Sperry (6) ; total N, by wet digestion with sulfuric acid, potassium sulfate, and Cu followed by nesslerization of the digest (7).

Estimation of XS has been estimated by a modification of the Carius method in which aqua regia is used as a digesting agent instead of nitric acid. This modification was introduced because it was found that methi-

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onine is only partly digested by nitric acid and is completely digested by aqua regia. SO,= in the d igest is titrated as benzidine sulfate.

Testing Materials for Digestibility by Trypsin-The material to be tested is treated with 4 per cent its weight of 1:300 trypsin at pH 9.0 at 37” for 2 weeks. Trypsin is added in two portions, one at the beginning and one on the 7th day. Toluene is used to prevent bacterial infection. At the end of the 2 week period, the whole digest is used for NH2-N estimation. Samples have always been run in duplicate; blanks have been run with trypsin alone.

Estimation of Protein Present in Lipide Extracts-The amount of protein present in lipide extracts can be estimated directly by separation of the protein residue as described below, or, indirectly by computation from the amount of amino acids liberated by acid hydrolysis of the protein present. The lipide material is analyzed for a-amino acid N as described elsewhere (8). The value obtained represents mainly phosphatidyl serine. Then, a sample of material is hydrolyzed with 6 N HCl at 100” overnight. The hydrolysate is neutralized to brom phenol blue by successive dropwise addition of 18 N NaOH, 1 N HCI, and 0.2 N NaOH. Aliquots are taken for a-amino acid N analysis by the ninhydrin method of Van Slyke et al. (9). The amount of protein present in the material analyzed can be com- puted from the formula, per cent a-amino acid N in the material after acid hydrolysis minus the per cent a-amino acid N in the intact material X 12. The factor 12 is the quotient of the ratio lOO/(S.S - 0.15) and is derived from the fact that the isolated protein contains 0.1 to 0.2 per cent (average 0.15 per cent) &-amino acid N before acid hydrolysis and 8.5 per cent after complete acid hydrolysis.

Presence of Proteins in Washed Lipide Extracts from Tissues

Chloroform-methanol extracts from tissues have been prepared by the method described in an accompanying paper (2). These washed extracts have been analyzed for proteins. In each case proteins present have been isolated by taking the washed extracts to dryness as described in the same paper (2). The insoluble protein residue has been collected on a filter paper and the material thus obtained has been analyzed for N, P, and for a-amino acid N before and after acid hydrolysis. These residues have been found to contain 11 to 12 per cent N, 0.2 to 0.4 per cent P, <0.2 per cent a-amino acid N before hydrolysis, and from 7 to 7.5 per cent a-amino acid N after acid hydrolysis. They give a strongly positive biuret test and are resistant to the a&on of trypsin. The following amounts have been found to be present in different tissues (expressed as mg. of proteo- lipide protein per gm. of wet tissue): brain white matter 20 to 25, brain gray matter 6.0, heart 3.48, kidney 1.95, liver 1.63, lung 0.94, smooth

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muscle (uterus) 0.61, and skeletal muscle (biceps) 0.40. Brain tumors ranged between white and gray matter in their proteolipide content. No proteolipides have been found in blood plasma.

Rudy of Proteolipide Protein from White Matter-From white matter, proteolipide protein has been isolated repeatedly, with a uniform yield of about 20 gm. of protein per kilo of fresh tissue. It is a residue insoluble in water and organic solvents which contains 12 per cent N, 0.3 to 0.4 per cent P, and 1.5 per cent S. On exhaustive extraction with boiling chloro- form-methanol, 2: 1 mixture by volume, the composition of the residue

TABLE I Composition of Protein Moiety from Proteolipides of White Matter of Brain

Constituents N in constituents ’ per cenT Of weight of origina protem

Leucine, isoleucine, phenylalanine. .... Methionine ............................ Tyrosine + valine. ................... Proline ................................ Alanine + glutamic acid. ............. Threonine ............................. Aspartic acid ...... Serine ............. Glycine. .......... Arginine. ......... Lysine ............ Histidine. ......... Cystine. .......... Ammonia. ........

. .

. .

......

......

...... .

. . . . . .

......

......

......

......

2.49 0.36 1.01 0.28 1.81 0.84 0.43 0.71 1.14 1.01 0.91 0.64 0.45 0.89

Total................................................. 12.97

changes to 14 per cent N, <O.l per cent P, and 1.76 per cent S. This appears to represent the proteolipide protein free of any significant amount of lipide contaminants.

The residue is completely insoluble in water; it dissolves incompletely in N NaOH at room temperature, heating on a steam bath being necessary for complete solution. On analysis it is found to contain 0.1 to 0.2 per cent a-amino acid N and about the same concentration of NHZ-N. On hydrolysis by 6 N HCl at 100” the values for a-amino acid N increase with length of time of hydrolysis, a value of 8.5 per cent a-amino acid N being reached after 8 hours and no significant increase being obtained after that time. In solution in alkali the material gives a strongly positive biuret test, but, owing to the fact already mentioned that it is necessary to use hot alkali to insure complete solution of the protein, the quantitative

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biuret method for estimation of proteins (10) does not yield reproducible values. Table I gives an amino acid analysis on proteolipide protein from white matter for which we are indebted to Dr. S. Moore and Dr. W. II. Stein. It can be seen that 92.7 per cent of the N in this material has been accounted for. The balance probably includes tryptophan, which is de- stroyed during hydrolysis.

Isolation of Proteolipides from Brain White Matter

Extraction of Tissue and Separation from Extract of Fluff and, of Chloro- form Phase-Unless otherwise stated, the procedure was carried out at 4”. Two 30 gm. batches of cattle white matter were each extracted with 600 cc. of chloroform-methanol mixture, 2: 1 by volume, by homogenizing for 2 minutes in the Waring blendor. The two homogenates were combined and filtered. 1100 cc. of filtrate were obtained. A 1 liter Erlenmeyer flask was filled to the brim with extract and completely immersed in a 5 gallon enamel porcelain pot nine-tenths filled with water. The mouth of the flask was 2 inches below the surface of the water. After standing for 48 hours the Erlenmeyer flask was taken out of the water. It contained a lower transparent chloroform phase, an upper water phase, and a fluff at the interphase. As much water as possible was removed without disturb- ing the fluff and the flask was placed at -10”. After 6 hours the fluff had frozen, while the chloroform remained liquid. Fluff and chloroform were separated by filtration through a filter paper and funnel precooled to -10”.

Processing of Flu$---The fluff on the filter paper and some that had remained on the walls of the flask were transferred to a 100 cc. centrifuge tube and centrifuged at 4” in an angle centrifuge at 4500 r.p.m. for 30 minutes. At the end of the centrifuge run a certain amount of chloroform that had collected at the bottom of t,he tube was removed by means of a siphon. The centrifugation was repeated and some additional chloroform that had collected was removed. The procedure was repeated; no more chloroform collected at the bottom of the centrifuge tube. The contents of the tube were transferred to a 50 cc. aluminum centrifuge tube and centrifuged at 40,000 X g (15,000 r.p.m. with a 9 inch rotor) for 1 hour. After centrifugation the contents of the tube had separated into a water- clear supernatant and a precipitate. The supernatant was decanted and the precipitate was frozen by placing the tube at -10”. The frozen pre- cipitate was transferred to a 250 cc. beaker at 4” and to it were added 50 cc. of chloroform and 25 cc. of methanol. The mixture was stirred. As the frozen precipitate thawed out, it contributed water to the system and soon two phases separated, giving the mixture a milky appearance. Stirring was continued and meanwhile methanol was added dropwise at

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a rate of 3 or 4 cc. a minute until a one-phase system resulted, as shown by the mixture becoming transparent.

Isolation of Proteolipide A-The solution of fluff was transferred to a 100 cc. glass-stoppered cylinder. It had a volume of 90 cc. 10 cc. of methanol were added to the solution, the cylinder was stoppered, its con- tents mixed by repeated inversion, and the cylinder placed at - 10’. After 40 hours, a precipitate that had formed was collected on a filter paper and funnel precooled to -10”. The filter paper holding the precipitate was dried in a vacuum desiccator at room temperature to constant weight. 1.2 gm. of a white powder were obtained (proteolipide A).

Isolation of Proteolipide B-To the filtrate from proteolipide A was added an equal volume of acetone, and, after mixing, the solution was allowed to stand at 4” overnight. A precipitate that had formed was collected by centrifugation and dried in a vacuum desiccator at room temperature to constant weight. 0.6 gm. of a white powder was obtained (proteo- lipide B).

Isolation of Proteolipide C-The chloroform phase, which had a volume of about 600 cc., was placed in a 1 liter heavy walled, round bottom flask and the solvent removed by vacuum distillation. To the residue in the flask were added 200 cc. of acetone; the acetone was brought to boiling by placing the flask in a water bath at 70”, the flask was removed from the bath, and the residue then carefully comminuted by means of a glass rod so as to insure intimate contact with the acetone. The flask was stop- pered and allowed to stand at 22’ overnight. The next morning most of a clear supernatant could be decanted. The remaining acetone was then removed by vacuum distillation. To the residue were added 250 cc. of ether, which was brought to boiling. After thorough mixing of the resi- due with the ether, the flask was stoppered and placed at 4” overnight. The next day most of a clear supernatant could be decanted. The re- maining ether and the residue were separated by centrifugation at 2500 r.p.m. for 30 minutes. After decanting the clear ether supernatant, the residue was dried in a vacuum. The dry residue was then dissolved in 10 cc. of chloroform and to the chloroform solution were added 400 cc. of ethanol. The solution was mixed and allowed to stand for 2 hours at 22”. A precipitate formed. It was collected and dried in a vacuum desiccator. 0.65 gm. of a white powder was obtained (proteolipide C).

General Information on Procedure o j Isolation of Proteolipides-The pro- cedure as described above has been repeated eight times with uniform yields of uniform products. The procedure must be run as rapidly as possible; undue delay results in the separation of partly insoluble proteo- lipides or of proteolipide B that cannot be recrystallized. The procedure may have to be varied on the two following points. Sometimes two cen-

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trifugations of the fluff at 4500 r.p.m. do not suffice to remove all of the chloroform from the fluff. Thus, centrifugation should be repeated for as long as chloroform collects at the bottom of the tube. Sometimes in redissolving the fluff some material remains undissolved. In this case chloroform should be added until a milky appearance of the solution shows the separation of phases. After stirring for 30 seconds, enough methanol is added to form one single phase. Usually this results in complete dis- solution of the fluff.

The composition of the fluff prepared as described is quite constant. It represents 30 per cent of the solutes in the original tissue extract. Its solids contain 3.9 to 4.3 per cent N, 10 to 11 per cent carbohydrate (as galactose), and 1.1 to 1.3 per cent P. It consists of about 20 per cent pro- tein, 30 per cent phosphatides, and 50 per cent cerebrosides.

The solids in the chloroform phase account for about 62 per cent of the solids in the original extract. They consist of 8 to 9 per cent protein, 30 to 35 per cent cholesterol, 5 to 10 per cent cerebrosides, and about 50 per cent phosphatides. In the process of isolation of proteolipide C from the solutes in the chloroform phase, acetone removes about 36 per cent of the solids, mostly cholesterol. Ether removes about 30 per cent of the solids, mostly phosphatides. Ethanol removes about 25 per cent, a mixture of phosphatides and cerebrosides. The yield of proteolipide C is about 8 to 10 per cent of solids in the chloroform phase.

Behavior of Proteolipides on Equilibration between Chloroform-Methanol White Matter Extracts and Water-The distribution of proteolipides be- tween the chloroform phase and fluff after equilibration of the chloroform- methanol tissue extract with at least 5-fold its volume of water is affected by at least two factors. One appears to be the ratio, area of interphase to volume of extract. Thus, if 100 cc. of white matter extract are placed in a 100 cc. beaker and the beaker submerged in 1 liter of water, when equilibrium has been reached 80 to 90 per cent of the protein from the original extract is in the fluff, only 10 to 20 per cent remaining in the chloroform phase. When a 100 cc. aliquot of the same extract is placed in a 100 cc. cylinder and the cylinder immersed in 1 liter of water, only 15 to 30 per cent of the protein is found in the fluff, 70 to 85 per cent remain- ing in the chloroform. Another factor that affects the distribution is the time of contact between extract and water, after equilibrium has been reached, Thus, if three 2 liter Erlenmeyer flasks are filled to the brim with chloroform-methanol extract of white matter and submerged in 50 li- ters of water, and the fluff in the different flasks is collected after 2, 4, and 6 days, respectively, it is found that the fluff contains 49 per cent of the proteins after 2 days, 53.5 per cent after 4 days, and 58 per cent after 6 days.

Composition of Proteolipide A-This material is a birefringent white

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powder. N 3.8 to 4.3 per cent, carbohydrate (as galactose) 14 per cent, and 1’ 0.2 to 0.4 per cent. It consists of 20 per cent protein, 65 to 75 per cent cerebrosides, and 5 to 15 per cent phosphatides. It can be dissolved in chloroform-methanol-water mixtures by the following procedure. The powder is suspended in a small volume of water and to the suspension is added IO-fold its volume of chloroform. The mixture is shaken vigor- ously. It can be seen that the powder swells and loses some of its solid character. Soon it can be seen that no solid dry particles remain. Enough methanol is then added slowly to the mixture to bring water and chloro- form into one single phase with methanol. If some material remains un- dissolved, chloroform is added dropxvise until the system becomes hiphasic (as sholvn by a milky appearance). The mixture is shaken for a short time and one phase reformed by dropwise addition of methanol. Solu- tions up to 10 per cent concentration can be prepared by this method.

Proteolipide A is obviously a mixture of proteolipides and of fret lipides. I{y many different procedures it is possible to separate from it pure lipide fractions without splitting the proteolipide. The constituent> prot,ein can be split) from the lipidc by drying solutions prepared as described. The isolated protein moiety contains 1.76 per cent S. It is trypsin-resistant.

Composition oj I’roteolipide B-This preparation is a microcrystalline white powder. It consists entirely of well formed rosettes or sphcrules (Fig. 1). N 8.2 per cent, P 1.30 per cent, carbohydrate (as galactose) 4.0 per cent. It contains about 50 per cent protein, 20 per cent cerebro- sides, and 30 per cent phosphatides. It can be dissolved in chloroform- methanol-water mixtures by the procedure described for proteolipidc A. Solutions up to a 10 per cent concentration have been prepared. Drying the solution results in separating the constituent protein from the const,i- tuent lipides. The protein thus obtained contains 1.76 per cent S and is trypsin-resistant. From this solution, proteolipide B can be recrystal- lized by addition of an equal volume of acetone to the solution. The composition of the crystals is the same as that of the original proteolip- idc B.

In spite of t’he fact that these crystals have been obtained eight different times with the same composition, and that their composition does not change on recrystallization, the authors make no claim that proteolipide B is a pure substance. The reason for this reservation is 2-fold. On one hand, it is felt that crystallinity alone is not sufficient proof of the purity of a substance such as a proteolipide, about the behavior and properties of which much remains to be learned. On the other hand, by introducing slight changes in the procedure of isolation described, it is possible to obt.ain cryst,als that have a different composition than that reported here. The composition of these crystals does not change on recrystallization. In preference to claiming that a number of different pure subst,ances have

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816 PROTEOLIPIDES

been isolated, the authors feel that maybe what is being dealt with is a phenomenon of isomorphic crystallization of one or more proteolipides with cerebrosides.

Composition of Proteolipide C-This mat.erial is obtained as a birefrin- gent white powder. N 10.5 to 11 per cent, P 1.30 per cent, carbohydrate (as galactose) 1 per cent. It consists of 70 to 75 per cent protein, the balance being mainly phosphatides. It is soluble in chloroform and in chloroform-methanol mixtures without addition of water. These solutions can be dried without splitting the bond between the constituent lipides

FIG. 1. Photomicrograph of proteolipide I3 in polarized light. Magnification 80 x.

and proteins. However, if 5 per cent water is added to a solution of pro- teolipide C in a chloroform-methanol mixture and the solution dried, the bond between the lipide and protein moieties is split, although about 25 per cent of the protein present still remains soluble in the chloroform. This suggests the possibility that there is in proteolipide C a proteolipide fraction that is not split by drying from solutions in chloroform-methanol- water mixtures. The protein moiety isolated from proteolipide C con- tains 1.76 per cent S and is trypsin-resistant.

SUMMARY

1. Tissues contain substances to which the name of proteolipides has been given. These are lipoproteins which exhibit solubilities quite dif-

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ferent from the solubilities of other known lipoproteins. Thus, while the latter are usually soluble in water or dilute salt solutions, proteolipides are insoluble in water and freely soluble in chloroform-methanol-water mixtures.

2. Proteolipides are present in the following tissues that have been stud- ied and which are listed in order of decreasing content of proteolipides: brain white matter, brain tumors, brain gray matter, heart, kidney, liver, lung, smooth muscle, and skeletal muscle. They are absent from blood plasma.

3. From white matter three different proteolipide fractions have been isolated. One of them has been consistently obtained as a crystalline compound. The other two are birefringent powders. All three are insol- uble in water and soluble in chloroform-methanol-water mixtures. Their protein moieties contain 1.76 per cent S and are resistant to the action of trypsin.

BIBLIOGRAPHY

1. Folch, J., and Lees, M., Federation Proc., 9, 171 (1950). 2. Folch, J., Ascoli, I., Lees, M., Meath, J. A., and LeBaron, F. N., J. Biol. Chem.,

191, 833 (1951). 3. Schmidt, G., Benotti, J., Hershman, B., and Thannhauser, S. J., J. Biol. Chem.,

166,505 (1946). 4. Page, I., Chemistry of the brain, Baltimore, 203 (1937). 5. Folch, J., J. Biol. Chem., 1’77, 505 (1949). 6. Sperry, W. M., Ind. and Eng. Chem., Anal. Ed., 14,88 (1942). 7. Peters, J. P., and Van Slyke, D. D., Quantitative clinical chemistry; Methods,

Baltimore, 532 (1932). 8. Folch, J., J. Biol. Chem., 174, 439 (1948). 9. Van Slyke, D. D., Dillon, R. T., MacFadyen, D. A., and Hamilton, P., J. Biol.

Chem., 141, 627 (1941). 10. Hiller, A., Greif, R. L., and Beckman, W. W., J. Biol. Chem., 176, 1421 (1948).

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Jordi Folch and M. LeesISOLATION FROM BRAIN

TISSUE LIPOPROTEINS: THEIR PROTEOLIPIDES, A NEW TYPE OF

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