ARTICLE:
`A SIMPLE METHOD FOR THE
`ISOLATION AND PURIFICATION OF
`TOTAL LIPIDES FROM ANIMAL
`TISSUES
`(cid:160)
`Jordi Folch, M. Lees and G. H. Sloane Stanley
`1957, 226:497-509.
`
`J. Biol. Chem.(cid:160)(cid:160)
`
`Access the most updated version of this article at
`
` http://www.jbc.org/content/226/1/497.citation
`
`Find articles, minireviews, Reflections and
`JBC Affinity
`Classics on similar topics on the
`.
`Sites
`(cid:160)
`Alerts:
`When this article is cited
`•(cid:160)
`
` When a correction for this article is posted
`•(cid:160)
`(cid:160)
`Click here
`alerts
`
`
`
` to choose from all of JBC's e-mail
`
`This article cites 0 references, 0 of which can be
`accessed free at
`http://www.jbc.org/content/226/1/497.citation.full.h
`
`tml#ref-list-1
`(cid:160)
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on June 12, 2013
`
`000001
`
`(cid:160)
`

`
`A SIMPLE METHOD
`OF TOTAL
`
`FOR THE
`LIPIDES
`
`ISOLATION
`FROM
`ANIMAL
`
`AND PURIFICATION
`TISSUES*
`
`(From
`
`AND G. H. SLOANE STANLEY1
`BY JORDI FOLCH, M. LEES,t
`Research Laboratories,
`and
`Waverley,
`the Department
`the McLean
`Hospital
`of Biological
`Chemistry,
`Harvard
`Medical
`School,
`Boston,
`Massachusetts)
`
`(Received
`
`for publication,
`
`August 23, 1956)
`
`Work from this laboratory resulted in the development of a method for
`the preparation and purification of brain lipides (1) which involved
`two
`successive operations.
`In the first step, the lipides were extracted by
`homogenizing the tissue with 2: 1 chloroform-methanol (v/v), and filtering
`the homogenate.
`In the second step, the filtrate, which contained the
`tissue lipides accompanied by non-lipide substances, was freed from these
`substances by being placed in contact with at least 5-fold its volume of
`water. This water washing entailed the loss of about 1 per cent of the
`brain lipides.
`This paper describes a simplified version of the method and reports the
`results of a study of its application to different tissues, including the effi-
`ciency of the washing procedure in terms of the removal from tissue lipides
`It also re-
`of some non-lipide substances of special biochemical interest.
`ports some pertinent ancillary
`findings. The modifications
`introduced
`into the method pertain only to the washing procedure. A chloroform-
`methanol extract of the tissue, prepared as described in the original version
`of the method, is mixed with 0.2 its volume of water to which, for certain
`purposes, different mineral salts may be added. A biphasic system with-
`out any interfacial fluff is obtained (2). The upper phase contains all of
`the non-lipide substances, most of the strandin, and only negligible amounts
`of the other lipides. The lower phase contains essentially all the tissue
`In comparison with the original method, the
`lipides other than strandin.
`present version has the advantage of being simpler, of being applicable to
`any scale desired, of substantially decreasing the losses of lipides incidental
`to the washing process, and, finally, of yielding a washed extract which
`can be taken to dryness without
`foaming and without splitting of the
`proteolipides (3).
`* This work has been aided by grant No. B-130
`Health Service.
`t Fellow of the American Cancer Society, 1951-53.
`$ Eli Lilly Traveling
`Fellow
`in Medicine,
`1953-55.
`497
`
`the United States Public
`
`from
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on June 12, 2013
`
`000002
`
`

`
`498
`
`ISOLATION
`
`OF
`
`TOTAL
`
`TISSUE
`
`LIPIDES
`
`Procedure
`
`in lip-
`poor
`relatively
`tissues
`and methanol
`the chloroform
`
`Reagents-
`Reagent grade.
`Chloroform.
`For use with
`Reagent grade.
`Methanol.
`ides, such as muscle or blood plasma, both
`must be redistilled.
`2 : 1 by volume.
`Chloroform-methanol mixture.
`Pure solvents upper phase and pure solvenk
`lower phase. Chloroform,
`methanol, and water are mixed
`in a separatory
`funnel
`in the proportions
`8: 4: 3 by volume. When
`the mixture
`is allowed
`to stand, a biphasic sys-
`tem is obtained.
`The two phases are collected separately
`and stored
`in
`glass bottles.
`It has been found that the approximate
`proportionBof
`chloro-
`form, methanol, and water
`in
`the upper phase are 3: 48: 47 by volume.
`are 86: 14: 1. Either of the
`In the lower phase,
`the respective proportions
`phases may be prepared directly by making use of the above, proportions,
`0..02 pm cent CaCl2, 0.017 per- cent
`Pure solvents upper phase containing
`2wgCZ~ 0.99 per cent NaCE, or 0.37 per cent KCZ. These solutions can be
`prepared in one of two ways. One is to shake the appropriate amount of
`salt with pure solvents upper phase in 8 glass-stuppered vessel until solufiion
`is complete. The other is to proceed a8 for the preparation of pure solvents
`upper and lower phases except that, instead of water, 0.04 per cent aqueous
`CaClz, 0.034 per cent aqueous MgClz, 0.58 per cent aqueous NaCl,. or 0.74
`per cent aqueous KC1 is used.
`Extraction of Lilpides-For
`the purposes of this description, the volume
`of a tissue sample will be computed on the assumption that the tissue has
`the specific gravity of water; i.e., the volume of 1 gm. of tissue is 1 ml. The
`tissue or tissue fraction is homogenized with 2: 1 chloroform-methanol mix-
`ture (v/v)
`to a final dilution 2O-fold the volume of the tissue sample-; i.e.,
`the homogenate from 1 gm. of tissue should be diluted to a volume of 20
`ml. For amounts of tissue up to 1 gm., the homogenization is carried out
`in a Potter-Elvehjem
`type of homogenizer, the tube of which has been
`weighed, and calibrated at the volume of the final dilution of the particular
`tissue homogenate. Thus, the tissue sample can be weighed and the ho-
`mogenate diluted to volume without a transfer. For brain or tissues of
`similar consistency, 3 minutes suffice fur complete homogenization.
`Tougher tissues will require lengthier homogenization, and some organs
`rich in connective tissue, e.g. peripheral nerve, may require special handling
`such as grinding with a mortar and pestle at the temperature of dry ice
`before homogenization with
`the solvent mixture.
`For amounts greater
`than 1 gm., the tissue is homogenized in an adequate blendor with about
`a 17-fold volume of solvent mixture;
`the balance of solvent mixture re-
`quired to dilute the humogenate to final volume is used to insure the quac
`titative
`transfer of the homogenate into a volumetric
`flask. After
`tem-
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on June 12, 2013
`
`000003
`
`

`
`J.
`
`FOLCH,
`
`M.
`
`LEES,
`
`AND
`
`G. H.
`
`SLOANE
`
`STANLEY
`
`499
`
`is
`the homogenate
`final volume adjustment’,
`and
`equilibration
`perature
`the
`vessel.
`For
`paper
`into a glass-stoppered
`through
`a fat-free
`filtered
`this extract
`corresponds
`to 0.05 its volume of
`purposes of computation,
`corresponds
`to 0.05 gm. of tissue.
`tissue;
`i.e., 1 ml. of extract
`with
`Washing of Crude Extract-The
`crude extract
`is mixed
`thoroughly
`0.2 its volume of either water or an adequate salt solution
`(see “Experi-
`mental”),
`and the mixture
`is allowed
`to separate
`into
`two phases, without
`interfacial
`fluff, either by standing or by centrifugation.
`The volumes of
`the upper and lower phases are, respectively,
`40 and 60 per cent of the total
`volume of the system.
`As much of the upper phase as possible
`is removed
`by siphoning, and removal of its solutes
`is completed by rinsing
`the inter-
`face three
`times with
`small amounts of pure solvents upper phase
`in such
`a way as not to disturb
`the lower phase.
`Finally,
`the
`lower phase and
`remaining
`rinsing
`fluid are made into one phase by the addition of meth-
`anol, and the resulting
`solution
`is diluted
`to any desired
`final volume by
`the addition of 2: 1 chloroform-methanol
`mixture.
`feasi-
`technically
`The procedure can be run on any scale that
`is otherwise
`ble, and the actual details of operation will vary according
`to the amount
`of extract being washed.
`For instance,
`if 10 ml. of crude extract are to be
`washed,
`the extract
`is placed in a 15 ml. centrifuge
`tube.
`To it are added
`2 ml. of either water or salt solution,
`the two
`liquids are mixed with a stir-
`ring rod, the rod is then rinsed
`into the tube with a minimal amount of pure
`solvents
`lower phase, and the tube is capped with aluminum
`foil and centri-
`fuged until complete separation of the syst.em int,o two phases without
`any
`interfacial
`fluff
`is obtained.
`The duration
`of centrifugation
`varies
`from
`about 20 minutes at 2400 r.p.m.
`for white matter extracts
`to a very short
`time
`for blood plasma.
`The volumes of the upper and lower phases are
`4.8 and 7.2 ml., respectively.
`The upper phase is removed as completely
`as possible with a pipette or with a suction arrangement
`such as the one
`described by Van Slyke and Rieben
`(4). Next,
`the inside wall of the t,ube
`is riuscd with about 1.5 ml. of pure solvents upper phase, which are allowed
`to flow gent’ly
`from a pipette so t.hat the washing
`fluid collects on top of
`the lower phase without
`any mixing of the two phases.
`The tube
`is ro-
`tated gently
`to insure mixing of the rinsiug
`fluid with
`the remaining original
`upper phase, and the mixture
`is removed.
`This
`rinsing of the tube wall
`and interphase with pure solvents upper phase is repeated
`twice.
`Finally,
`the lower phase is diluted
`to a volume of 10 ml. as outlined above. With
`tissues poor in proteolipides,
`e.g. muscle, plasma, and liver, or if time
`is no
`object, centrifugation may be omitted
`from
`t,he washing
`procedure.
`Ill-
`stead,
`the extract plus water mixture
`can be allowed
`to separate
`into
`two
`In that case, it is more convenient
`to carry
`phases by prolonged standing.
`out the \vashiug
`ill glass-stoppered
`cylinders.
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on June 12, 2013
`
`000004
`
`

`
`500
`
`ISOLATION
`
`OF
`
`TOTAL
`
`TISSUE
`
`LIPIDES
`
`if so indicated by the size and nature of the tissue
`changed in many details
`sample or by the particular
`problem under study.
`Thus,
`if necessary,
`in
`the preparation
`of the crude extract,
`the tissue homogenate
`can be diluted
`to more
`than 20-fold
`the volume of tissue.
`Also, centrifugation
`can be
`used in preference
`to
`filtration
`as a means of obtaining
`a clear extract.
`Centrifugation
`of the homogenate
`itself
`is unsatisfactory
`because
`the spe-
`cific gravity of the solvent mixture
`is too close to the density of t.he sus-
`pended material.
`Therefore,
`if centrifugation
`is to be used, it is necessary
`to lower
`the specific gravity of the homogenate by the addition of methanol.
`Usually,
`the addition of 0.2 its volume of methanol suffices
`for the purpose.
`The amount of methanol added must be noted.
`In the washing procedure described, chloroform, methanol, and water are
`present
`in the system
`tissue extract
`plus water
`in the proportions
`8:4: 3
`by volume, as can be computed
`if account
`is taken of the
`fact
`that
`the
`extract
`contains
`all the water
`from
`the
`tissue.
`These proportions
`are
`Therefore,
`in cases in which
`the tissue
`critical and must be kept constant.
`extraction
`has been substantially
`changed,
`it is necessary
`to modify
`the
`washing procedure
`in a way
`that will
`restore
`the required proportions
`of
`solvents.
`For
`instance,
`if the homogenate has been diluted
`to 40-fold
`the
`volume of tissue,
`the water
`contributed
`to the extract by the latter will be
`half as much as in the standard
`20-fold dilution;
`i.e., it will be 2 per cent
`of the extract as compared
`to the usual 4 per cent.
`Therefore,
`the amount
`of water added to the ext)ract
`for washing
`should be 22 per cent instead of
`the usual 20 per cent.
`If methanol has been added
`to the extract,
`twice
`as much chloroform must also be added and the amount of water adjusted
`accordingly.
`
`EXPERIMENTAL
`of the methods used in this study have been
`
`Analytical Methods-Most
`described elsewhere
`(3, 5).
`work had
`of Tissue Lipides-Earlier
`Degree of Completeness of Extraction
`removes all lipides
`from brain
`(1) and
`shown
`that
`the extraction
`procedure
`blood plasma
`(6), with
`the exception of a specific
`fraction of lipides which
`is combined
`to tissue proteins
`by a linkage which withstands
`the action
`of neutral solvents.
`In the present study,
`the completeness
`of extraction
`of lipides from
`liver and muscle was studied by reextracting
`the residue with
`hot solvent and determining
`the amount of lipides
`in the second extract.
`The original extraction
`can be considered
`complete
`if the second extract
`contains no more lipides
`than can be accounted
`for by the aliquot of first
`The experiment was carried out as fol-
`extract
`left wetting
`the residue.
`lows: The tissue was homogenized with
`chloroform-methanol
`as described,
`and the homogenate
`filtered
`through a previously weighed Riichner
`funnel,,
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on June 12, 2013
`
`000005
`
`

`
`J.
`
`FOLCH,
`
`Nl.
`
`LEES,
`
`AND
`
`C.
`
`H.
`
`SLOANE
`
`STANLEY
`
`501
`
`The
`residue became dry.
`insoluble
`the
`being stopped before
`filtration
`filter was weighed again and the weight of the wet residue
`it contained was
`computed by difference.
`Xcxt,
`the residue was
`reextracted
`with a new
`portion of solvent mixture by boiling under
`reflux
`for 24 hours,
`the sus-
`pension was
`filtered, and the twice extracted
`residue collected and dried
`to
`constant. weight,.
`The amount of first extract
`left wetting
`the tissue residue
`could
`then be computed
`from
`the equation, ml. of extract
`in residue =
`(weight of wet
`residue after
`first extraction minus weight of dried residue)/-
`In t’he case of liver, 40 gm. of tissue were
`(specific gravity of first extract).
`extracted as outlined above in succession with 760 ml. and 400 ml. of solvent
`mixture.
`The
`first extract
`contained
`2.46 mg. of lipides per ml., while
`28.8 ml. of extract with a comput,ed
`total
`lipide content of 71 mg. were
`left
`in the residue.
`The second extract
`contained a total of 69 mg. of lipides;
`i.e., the amount
`to be expected
`from
`the aliquot of the first extract
`in the
`residue.
`In an identical experiment with muscle
`tissue,
`the first extract
`contained 0.743 mg. of lipides per ml., while 24.3 ml. of extract with a total
`lipide content of 18.1 mg. were
`left wetting
`the residue.
`The second ex-
`tract contained a total of 21.6 mg. of lipides;
`i.e., 3.5 mg. more than were
`to be expected
`from
`the aliquot of the first extract
`remaining
`in the residue.
`This difference, which
`amounts
`to <0.5 per cent of total
`tissue
`lipides,
`cannot be considered significant.
`has been studied
`procedure
`washing
`Study of Washing Procedure-The
`by (a) determining
`the amount of lipides
`lost during
`the washing,
`(b) de-
`termining
`the amount of non-lipide
`subst.ances
`remaining
`in
`the
`lower
`phase, (c) investigating
`an effect of certain non-lipide
`substances
`upon the
`distribution
`of lipides between
`the two phases
`formed during
`the washing
`procedure,
`(d) determining
`the effect of mineral salts on the distribution
`of lipides in this particular
`biphasic system, and finally
`(e) ascertaining
`the
`efficiency of the washing
`procedure
`in relation
`to some substances
`of im-
`portance
`in metabolic
`studies by the use of radioisotopes.
`Loss of Lipides
`Incidental
`to Washing Procedure and Degree of Removal
`of Non-Lipide
`Contaminants-Since
`lipides are undialyzable,
`the amount
`of undialyzable
`substances
`in the upper phase would
`represent
`the maximal
`amount of lipides
`lost, and the dialyzable
`substances would,
`of necessity,
`In a typical experiment,
`175 ml. of
`represent non-lipide
`contaminants.
`brain white matter extract were washed with 35 ml. of water.
`The upper
`phase, which had a volume of 84 ml., was collected quantitatively.
`The
`lower phase was equilibrated with 84 ml. of pure solvents upper phase, and
`t,he resulting
`second upper phase was collected.
`Both upper phases were
`concentrated
`to dryness by vacuum distillation
`of the solvents,
`the residues
`were each dissolved
`in 10 ml. of water, and the solutions were dialyzed ex-
`haustively.
`The dialyzable and undialyzable
`fract’ions
`thus obtained were
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on June 12, 2013
`
`000006
`
`

`
`502
`
`ISOT~.4’1‘ION
`
`OF
`
`TOTAL
`
`TISSTJE
`
`IJPIDlCS
`
`fractions were com-
`in the undialyzable
`The solutes
`dried and analyzed.
`pletely soluble in chloroform-methanol,
`indicating
`that they were all lipides.
`The pertinent
`results are given
`in Table
`I.
`Thus,
`the values
`for the first
`upper phase show
`that no more than 0.3 per cent of the tissue
`lipides other
`than strandin was
`lost in the washing.
`Also, since the second upper phase
`cnontained only negligible amomlts of dialyzable
`substances,
`the conclusion
`is warranted
`that, after one washing,
`the
`lower phase
`is essentially
`free
`from non-lipide substances.
`The same type of experiment has been carried
`out wit,h white matter,
`gray matter,
`liver, and muscle, with
`the results
`given in Table II.
`In a.11 the t.issues studied, one washing was
`found suffi-
`
`Distribution
`
`of Solutes
`
`TABLE
`in CHC13:CH30H
`between
`Subsequent
`
`I
`
`Extract
`Fractions
`
`of Brain
`
`White Matter
`
`.................
`extract.
`in crude
`solutes
`1. Total
`...................
`solutes.
`2. 1st upper
`phase;
`total
`3. Dialyzable
`solutes
`...............................
`strandin).
`4. Undialyzable
`solutes
`(lipides
`+
`5. Strandin
`in undialyzable
`fraction
`................
`(5).
`-
`6. Lipides
`other
`than
`strandin
`(4)
`...........
`.................
`7. 2nd upper
`phase;
`total
`solutes.
`8. Dialyzable
`solutes.
`.............................
`9. Undialyzable
`solutes
`(lipides
`+
`10. Strandin
`in undialyzable
`fraction
`11. Lipides
`other
`than
`strandin
`(9)
`12. Final
`lower
`phase;
`total
`solutes
`13. Total
`lipides
`including
`strandin
`
`.......
`
`.......
`
`........
`
`strandin).
`................
`(10).
`-
`..................
`(4) +
`
`(9) +
`
`(12)
`
`Yield, mg.
`
`total
`as y.
`Yield
`in
`solutes
`crude
`extract
`
`2000.0
`95.85
`81.5
`14.35
`7.75
`6.6
`46.7
`1.7
`45.0
`4.6
`40.4
`1855.0
`1914.4
`
`4.79
`4.07
`0.72
`0.39
`0.33
`2.33
`0.08
`2.25
`0.23
`2.02
`92.75
`95.72
`
`cient for removing all the non-lipide contaminants from the crude extract.
`In the case of gray matter, lipides other than strandin lost in the course of
`the first washing amounted to no more than 0.6 per cent of the tissue lip-
`ides; for liver and muscle, the values were somewhat higher, ranging up to
`2 per cent.
`can be seen from
`Recognition of Lipide Distribution-Altering Factor-It
`Table II
`that the second upper phases contained more lipides than the
`corresponding first upper phases. This unexpected finding was investi-
`gated by preparing in duplicate six successive upper phases from aliquot,s
`of white and gray matter extracts, as described above. The lipides from
`each phase were recovered and analyzed (Table III).
`It was found in
`both cases that the amount of lipides increased markedly from the first to
`the second upper phase; it remained unchanged from the second to the
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on June 12, 2013
`
`000007
`
`

`
`J.
`
`FOLCH,
`
`M.
`
`LEES,
`
`AND
`
`G. H.
`
`SLOANE
`
`STANLEY
`
`503
`
`the sixth by a fairly con-
`through
`the third
`from
`third, and then decreased
`to the distribution
`of a group of lipides
`stant
`factor which
`corresponded
`exhibiting a distribution
`coefficient of about 2.7 in favor of the lower phase.
`The negligible amount of lipides in the first upper phase could be explained
`“distribution
`coefficient altering”
`only by assuming
`that some unknown
`factor had been in operation
`in the washing
`of the original extract.
`The
`
`Lipide
`
`and Non-Lipide
`Total
`
`II
`TABLE
`by First
`Removed
`Substances
`Lipide
`Extracts
`of Various
`
`and Second Washings
`Tissues
`
`of
`
`Tissue
`
`White
`
`matter
`
`Gray
`
`matter
`
`Liver
`
`Muscle
`
`2nd upper
`-
`
`phase
`
`Lipides
`
`other
`strandin
`
`than
`
`per C&
`tissue
`Zigides
`1.85
`1.58
`2.11
`2.18
`1.82
`2.06
`1.19
`1.34
`4.3s*
`3.92*
`3.25*
`3.25+
`2.08*
`2.41*
`
`4.1
`3.5
`4.6
`4.7
`1.5
`1.7
`0.8
`0.9
`1.9*
`1.7*
`1.6*
`1.6*
`0.31*
`0.36*
`
`-
`
`ran-lipid<
`ubstances
`&lyzabh
`solutes)
`
`mg. per
`m.
`fresh
`2issue
`0.27
`0.55
`0.19
`0.18
`0.35
`0.27
`0.11
`0.05
`0.04
`0.11
`0.41
`0.39
`0.13
`0.17
`
`1st upper
`
`phase
`
`Non-lipide
`substances
`(dialyzable
`solutes)
`
`Lipides
`
`other
`strandin
`
`than
`
`nrg. per gm.
`jresh
`tissue
`
`nrg. per gm.
`fresh
`tissue
`
`8.3
`7.8
`9.3
`9.2
`10.3
`10.2
`11.4
`11.9
`
`19.0
`18.8
`13.6
`13.9
`
`0.70
`0.51
`0.74
`0.69
`0.47
`0.47
`0.21
`0.44
`0.62
`0.54
`1.02*
`1.17*
`0.26*
`0.28*
`
`-
`
`0.31
`0.24
`0.34
`0.32
`0.56
`0.57
`0.31
`0.65
`1.4
`1.2
`2.0*
`2.4*
`1.P
`1.9*
`
`* Lipides,
`
`including
`
`strandin.
`
`in the second equilibration, most, likely
`effect of this factor was still evident
`because of contamination
`of the system by
`first upper phase.
`The lipides from white matter upper phases 3 through 6 were pooled and
`analyzed,
`in per cent: S 1.4, P 1.9, N 1.5, NH2-N 0.54, a-amino acid K 0.54,
`carbohydrate,
`as galact,ose, 7.9, S + P/K
`atomic
`ratio 0.98, atoms S pel
`moles of galactose 1.00, choline none.
`Thus,
`the
`lipides affected by
`the
`distribution-altering
`factor consisted of a mixture of 40 per cent sulfatides,
`35 per cent phosphatidyl
`serine, and 25 per cent other phosphatides;
`i.e.,
`they were mainly,
`if not exclusively,
`acidic
`lipides.
`facts
`observed
`Identification of Lipide Distribution-Altering
`Factors-The
`might he explained by assuming
`that
`the distribution
`of water,
`chloroform,
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on June 12, 2013
`
`000008
`
`

`
`504
`
`ISOLATION
`
`OF
`
`TOTAL
`
`TISSUE
`
`LIPIDES
`
`between
`the two phases had changed significantly
`and methanol between
`the first and subsequent equilibrations.
`This possibilit’y was
`investigated
`by determining
`the volume and the specific gravity of both phases through
`the procedure of preparation
`of six upper phases
`(see above).
`No changes
`were observed.
`Thus,
`it can safely be assumed
`that
`the composition
`of
`the phases had remained essentially
`constant.
`
`Lipides
`
`TABLE
`in Successive
`Upper
`Present
`CHCla:CH,OH
`Extract
`Pltts
`
`III
`
`of System
`Phases
`35 Ml. of Wale?
`
`175 Ml.
`
`of
`
`Upper phases
`
`lipides other
`White matter
`strandin
`
`than
`
`I
`I-
`
`Gray matter
`
`lipides other
`strandin
`
`than
`
`Yield
`
`P content
`
`Yield
`
`mg. *ET 175 ml.
`tissue
`ertract
`6.1
`4.5
`35.8
`30.1
`38.5t
`33.2t
`25.6t
`24.81
`20.01
`1s.ot
`11.ot
`13.0t
`
`per cent
`2.32
`2.82
`2.42
`2.69
`1.67
`1.9s
`2.01
`2.01
`2.06
`2.14
`2.10
`1.98
`
`4.1
`4.1
`13.2
`14.7
`13.4t
`12.4t
`9.ot
`9.1t
`7.7t
`9.3t
`3.4t
`5.1t
`
`Ia*
`lb*
`2a
`213
`3s
`3b
`4a
`‘lb
`-,
`8;1
`6a
`6b
`
`P content
`
`per telz t
`2.72
`2.96
`2.54
`2.54
`2.36
`2.50
`2.61
`2.67
`2.47
`2.54
`2.40
`2.70
`
`* Upper
`t Strandin
`values
`given
`are negligible.
`
`a and b refer
`phases
`estimations
`were
`for
`total
`lipides
`
`to duplicate
`not
`carried
`include
`strandin,
`
`experiments.
`out
`on
`lipides
`but
`the
`
`from
`amounts
`
`phases.
`these
`of strandin
`
`Thus
`present
`
`factor was one or more of the
`the
`could be that
`Another explanation
`solutes
`in the crude extract which would be removed by the washing
`pro-
`cedure, and therefore would be found
`in the first upper phase.
`This was
`shown
`to be the case by the
`following
`type of experiment.
`A stock of
`lower phase was prepared by washing
`crude white matter
`extract
`once
`with water.
`The solutes
`from
`the upper phase were
`recovered.
`Identical
`aliquots of lower phase were mixed with equal volumes of pure solvents
`upper phase. Different
`amounts
`of first upper phase solutes were added
`to some of the mixtures.
`After
`centrifugation,
`the upper phases were
`analyzed
`for P content, which had been shown
`to be a reliable
`indicator of
`the total amount of lipides present.
`It was
`found
`that
`the amount
`of
`lipides
`in the upper phases was decreased by
`the presence of the added
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on June 12, 2013
`
`000009
`
`

`
`J. FOLCH,
`
`M.
`
`LEES,
`
`AND
`
`G. H.
`
`SLOANE
`
`STANLEY
`
`505
`
`l), an
`(Fig.
`of the concentration
`logarithm
`to the
`in proportion
`solutes
`observation which provided a means for measuring
`the distribution-altering
`effect of any material.
`Thus,
`it was possible
`to trace
`this effect quantita-
`tively
`from
`the first upper phase solutes
`to their dialyzable
`fraction and to
`
`?I
`0
`
`’
`
`’
`’
`’
`’
`’
`’
`’
`80
`60
`40
`20
`IN AMOUNT
`% DECREASE
`OF LIPIDE
`IN UPPER PHASE
`FIG.
`1
`of solutes
`1. Effect
`FIG.
`extract
`chloroform-methanol
`phases
`two
`ides between
`the
`of different
`2. Effect
`FIG.
`phases
`of
`the
`same
`solvent
`matter
`chloroform-methanol
`A, CaC12.
`l
`, MgCL;
`
`’ 1
`100
`
`I
`
`I
`0
`
`I
`
`I
`
`100
`
`matter
`of
`lip-
`
`I
`I
`I
`60
`40
`20
`+
`IN AMOUNT
`% DECREASE
`OF LIPIDE
`IN UPPER PHASE
`FIG.
`2
`white
`the biphasic
`system
`of
`phase
`the upper
`from
`of water
`on
`distribution
`plus 0.2
`its volume
`the
`of a system
`of
`identical
`solvent
`composition.
`two
`the
`salts
`on
`the
`distribution
`of
`lipides
`between
`white
`composition
`as
`those
`obtained
`from
`the
`system
`extract
`plus
`0.2
`its volume
`of water;
`0, KCl;
`X, NaCl;
`
`i.e., the effect was caused by the mineral salts present
`
`the ash therefrom;
`in the crude extract.
`con-
`effect of different
`the distribution-altering
`By the same procedure,
`It
`centrations
`of NaCl, KCl, CaCL, and MgClz was determined
`(Fig. 2).
`was
`found
`that virtual absence of lipides
`from
`the upper phase could be
`obtained by the addition
`to it of CaClz or MgCh at a concentration
`of
`0.003 N, or of NaCl or KC1 at a concentration
`of 0.05 RT.
`Comparison between Amounts of Lipides Lost upon Washing with Water or
`with Mineral Salt Solutions-This
`comparison
`has been made by washing
`crude extracts
`of various
`tissues
`in parallel with either water or aqueous
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on June 12, 2013
`
`0000010
`
`

`
`506
`
`ISOLATION
`
`OF
`
`TOTAL
`
`TISSUE
`
`LIPIDES
`
`the
`and determining
`concentrations,
`salts at various
`solutions of different
`amount of lipides in the first upper phase.
`Table IV gives the comparative
`data for water and for 0.05 per cent CaClz which
`result
`in a concentration
`of Ca+
`in the upper phase of 3.8 m.eq. per liter.
`It can be seen that
`the
`use of the latter decreases
`the loss of lipides
`incidental
`to the washing.
`In
`cases in which
`the use of CaClz
`is contraindicated,
`as when an insoluble
`Ca salt might) be formed, a similar
`result’ can be obtained with 0.04 per cent
`
`TABLE
`of Lipides
`Rewloved
`Presence
`and Absence
`
`IV
`Phase
`Lower
`from
`of Added
`Ca++
`
`in
`
`ilttlomts
`
`-
`
`Lipides
`
`in 1st upper
`
`phase
`
`Tissue
`
`In absence
`
`of added
`
`Ca++
`
`expressed
`
`I
`
`as mg. per gm.
`
`fresh
`
`tissue
`
`In presence
`
`of added
`
`Cai+
`
`Strandin
`
`0.78
`0.74
`0.90
`0.91
`3.0
`2.7
`3.6
`0.19
`0.19
`
`White
`
`matter
`
`Gray
`
`matter
`
`Liver
`
`Muscle
`
`* Lipides
`
`including
`
`strandin.
`
`Lipides
`than
`
`other
`strandin
`
`Strandin
`
`Lipides
`than
`
`other
`strandin
`
`0.44
`0.41
`0.32
`0.33
`1.95
`1.15
`1.90
`0.12
`0.09
`
`0.70
`0.51
`0.74
`0.69
`0.47
`0.47
`0.38
`0.62
`0.54
`1.02*
`1.17*
`0.26*
`0.2P
`
`0.27
`0.28
`0.13
`0.18
`0.24
`0.13
`0.25
`0.28
`0.20
`0.52*
`0.54*
`0.14%
`0.04*
`
`is ex-
`
`The procedure
`
`MgC12, 0.73 per cent NaCl, or 0.88 per cent KCl.
`actly as described
`for water.
`is sufficient
`one washing
`Study of Effkiency
`of Repeated Washing-While
`in the case of metabolic
`to purify
`lipides
`for the usual analytical purposes,
`radioisotopes,
`it is often
`studies involving
`the use of substances
`labeled with
`necessary
`to free lipides
`from non-lipide
`contaminants
`possessing
`specific
`activities
`lOOO-fold or more that of the lipides.
`Such a degree of purifica-
`tion can be reached by equilibrating
`the lower phase repeatedly with por-
`tions of pure solvents upper phase containing
`salt.
`The procedure
`is as
`follows: The crude extract
`is washed with water or with an appropriate
`salt solution,
`as already described.
`After quantitative
`removal
`of the
`upper phase, a portion of pure solvent,s upper phase containing
`the appro-
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on June 12, 2013
`
`0000011
`
`

`
`J.
`
`FOLCH,
`
`M.
`
`LEES,
`
`AND
`
`G. H.
`
`SLOANE
`
`STANLEY
`
`507
`
`is capped.
`the tube
`and
`two phases are stirred,
`the
`is added,
`priate salt
`the upper phase
`is removed
`quantitatively.
`The
`After
`centrifugation,
`fresh portions
`of pure solvents upper phase containing
`equilibration with
`mineral salt is repeat,ed as many
`times as is indicated by ad hoc experiments
`of the type reported below.
`has been carried out in
`A study of the efficiency of repeated washings
`collaboration with Dr. Manfred Karnovsky
`of the Biophysics
`Laboratory
`of Harvard Medical School.
`2 mg. samples of a CY4- or P32-labeled com-
`pound were dissolved
`in 0.1 ml. of water
`and added
`t’o 10 ml. of a crude
`liver
`lipide extract.
`The level of activity
`ranged between 0.5 X
`IO6 and
`1.0 X 106 c.p.m. per 10 ml. of extract.
`The extract’s were washed
`repeat-
`edly as described above, aliquots of the lower phase were
`taken aft)er each
`
`Extent
`
`of Removal
`
`of Added
`
`Shstances
`
`by Repeated
`
`Il’ashing
`
`of Lipide
`
`Extract
`
`Labeled
`
`substance
`
`added
`
`Glycerol
`Glucose.
`Sodium
`Choline,
`“
`Serine
`Sodium
`
`......................
`.............
`.......
`.................
`acetate.
`.........
`added
`no C&l?
`CaClz
`added.
`...........
`.......
`...................
`phosphate,
`monobasic
`
`.....
`
`1st washing
`
`!
`
`4.3
`0.8
`1.1
`16.2
`2.4
`0.2
`0.26
`
`Amount
`
`remaining
`
`in
`
`lower phase
`
`after
`
`2nd washing
`
`/ 3rd washing
`
`1 4th washing
`
`Per cent original
`
`added
`
`radioactivity
`
`j
`
`0.7
`0.16
`0.12
`7.3
`0.2
`0.006
`0.08
`
`0.31
`0.07
`0.06
`2.0
`0.1
`0.007
`
`0.4
`0.09
`0.09
`4.5
`0.1
`0.007
`0.09
`
`-
`
`in the
`remaining
`test substance
`and the amount of radioactive
`washing,
`in a gas flow counter
`in the pro-
`lower phase was estimated by counting
`porbional
`range
`(7, 8). Glycerol,
`glucose, acetate,
`choline, serine, and
`phosphate have been studied
`in this way.
`It can be seen from Table V
`that, while
`the repeated washing procedure
`is highly effective,
`the rate of
`removal of the different
`substances
`in the successive mashings does not
`follow a theoretical
`decrement
`line, with
`the possible exception of serine
`and glycerol
`in the first] two washings.
`The difference
`in the behavior of
`choline in t,he presence and absence of added CaClz suggests
`that choline
`forms salts with acidic lipides
`in amounts determined by competition with
`other bases present.
`of strandin
`distribution
`in This Procedure-The
`Behavior of Xtrandin
`is affected by the addition of mineral salts
`to the
`between
`the two phases
`upper phase, especially by CaClz
`(Table
`IV).
`The effect of KC1 is much
`in the essential
`less marked, and even at KC1 concentrations
`that
`result
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on June 12, 2013
`
`0000012
`
`

`
`508
`
`ISOLATION
`
`OF
`
`TOTAL
`
`TISSUE
`
`LIPIDES
`
`is present
`the upper phase the bulk of strandin
`absence of acidic lipides from
`(9) on the
`in the
`first upper phase.
`The observations
`of Svennerholm
`likely
`in-
`effect of NaCl on the distribution
`of gangliosides, which most
`cluded strandin,
`suggest
`that
`the action of NaCl
`is similar
`to that of KCl.
`In summary,
`the use of KC1 or NaCl
`in this procedure affects
`the distribu-
`tion of strandin only slightly.
`To eliminate strandin
`from
`the lower phase
`completely,
`three washings with
`the appropriate
`salt solutions
`should suf-
`fice. To isolate strandin,
`the three washings
`are combined,
`concentrated
`almost
`to dryness,
`and dialyzed.
`Strandin will be found quantitatively
`in the undialyzable
`fraction.
`
`DISCUSSION
`the original procedure
`to modify
`started as an attempt
`The present work
`In a survey of possible alter-
`of washing
`crude lipide extracts with water.
`natives,
`crude brain white matter extract and water were mixed
`in var-
`ious proportions.
`Most mixtures
`resulted
`in emulsions which were hard
`to separate or were
`inseparable.
`The exception was a mixture
`obtained
`by adding
`to the extract 0.2 its volume of water, which, upon standing or
`by centrifugation,
`separated
`into
`two clear phases without
`the persistence
`of any interfacial
`fluff.
`Investigation
`of the two phases showed
`that
`the
`upper phase contained practically
`all of the non-lipide
`substances and only
`negligible amounts of lipides,