`Lipids
`
`ANJNI KOUL AND RAJENDRA PRASAD
`
`1 Background
`
`Lipids form a group of organic compounds which is widely
`distributed in living systems. The term lipid cannot be defined
`concisely, but it refers to a large number of compounds which
`have similar solubility properties (for more details on type of
`membrane lipids, the reader is referred to Chap. I, this Vol.).
`Membrane lipids are amphipathic molecules which are water(cid:173)
`insoluble and can be generally extracted from different sources
`by treatment with the combination of polar and non-polar
`organic solvents. The aim of the present chapter is to consider
`practical aspects of isolation oflipids. The chapter is intended
`to provide the reader with necessary information for the ex(cid:173)
`traction of membrane lipids with confidence. The extraction
`protocols described herein should provide the reader with suf(cid:173)
`ficient background to adopt the procedure to fit to the experi(cid:173)
`mental requirements.
`For the purpose of extraction, membrane lipids can be clas(cid:173)
`sified into three distinct groups:
`
`Non-Polar Lipids
`
`This group includes carotenoids, hydrocarbons, sterol esters,
`wax esters and glycerides. These lipids are often extractable
`
`School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067,
`India
`
`R. Prasad (ed.), Manual on Membrane Lipids
`© Springer-Verlag Berlin Heidelberg 1996
`
`Par Pharm., Inc.
`Exhibit 1008
`Page 001
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`38
`
`Extraction of Membrane Lipids
`
`with ether or chloroform, since they are weakly bound to each
`other and to the hydrophobic regions of the membrane by Van
`der Waals forces.
`
`Polar Lipids
`
`This group includes phosphoglycerides, glycolipids and sterols
`which are not only held by Van der Waals forces but are also
`bound by hydrogen bonds and, in some cases, by electrostatic
`interactions. These bonds are less readily disrupted and, there(cid:173)
`fore, lower alcohols or acetone must be used to free these lipids
`from membranes. Chloroform:methanol (2: 1, v/v) is com(cid:173)
`monly used as an extraction mixture which removes polar
`lipids from membranes. However, acidic phospholipids and
`polyphosphoinositides are not generally extractable by this
`combination of polar and non-polar solvents, one must con(cid:173)
`sider adding 0.25% HCl (cone.) to the solvent mixture for com(cid:173)
`plete extraction of these lipids. Since plasmalogens would be
`hydrolysed by the acid, the use of acid should be avoided if
`plasmalogens are under investigation (see Chap. VII, this Vol.
`for more details).
`
`Anchored Lipids
`
`This group includes fatty acids and hydroxy fatty acids bound
`covalently through ester, amide or glycosidic bonds, predomi(cid:173)
`nantly to polysaccharide structures. These fatty acids are ex(cid:173)
`tractable only after acidic or basic hydrolysis of the covalent
`bonds.
`Before the extraction of membrane lipids, it is imperative to
`know that one is dealing with desired pure membrane fraction
`(discussed in Chap. II, this Vol.). Membrane lipids from ani(cid:173)
`mal, plant and microorganism sources should ideally be ex(cid:173)
`tracted as soon as possible after their preparation to avoid
`
`Par Pharm., Inc.
`Exhibit 1008
`Page 002
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`Anjni Koul and Rajendra Prasad
`
`39
`
`changes in lipid components. When this is not possible, the
`membrane fraction should be frozen rapidly on dry ice and
`stored in a sealed glass container at -70°C in an atmosphere of
`nitrogen. Under these conditions, the sample can be kept for
`several months.
`
`2 Extraction Protocols of Membrane Lipids
`from Different Sources
`
`General Principles of Extraction. Folch and Stanley (1957)
`procedure of lipid extraction is the most common method
`used. However, modifications to this method have also
`been adopted. Twenty volumes of chloroform: methanol (2: 1,
`v/v) mixture per volume of tissue or membrane pellet or
`suspension is recommended to yield a single homogeneous
`suspension. If the ratio is small (12-15 volumes) two phases
`will be formed, more solvent would be required to obtain a
`single phase. The single phase of solvent provides better inter(cid:173)
`action of polar and non-polar solvents with membrane lipids.
`High ratio is probably harmless to some extent, but too high a
`ratio of solvent will lower the water content, making polar lipid
`extraction incomplete.
`There are several protocols of lipid extraction which
`differ slightly from each other. Membrane lipid extraction
`protocols commonly used for membranes derived from
`different sources are described below. These protocols can
`also be used for the extraction of total lipids from tissues of
`different origin.
`
`• Benzene
`• Chloroform
`• Methanol
`• Propan-2-ol
`• CaC12 anhydrous
`
`Reagents
`and
`solvents
`
`Par Pharm., Inc.
`Exhibit 1008
`Page 003
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`Extraction of Membrane Lipids
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`• 0.9% NaCl
`• Nitrogen gas
`
`Equipment • Thermal block or rotary evaporator
`
`Extraction 1.
`from red
`blood cells
`
`Resuspend the RBC membrane fraction pellet directly in
`glass tubes (with stoppers) in at least 20 volumes of
`chloroform:methanol (2: 1, v/v) or more commonly used
`alcohol: ether (3: 1, v/v). Shake the suspension vigorously
`for 2-3 min. (When using tissue for lipid extraction, it is
`necessary to homogenise the tissue extract in a glass
`homogeniser for uniform suspension.)
`
`Note. Normally 20 volumes of solvent mixture is sufficient
`to make a single homogeneous phase. If the suspension
`does not form a single phase, more solvent mixture can be
`added.
`
`2. Flush the suspension with N2 gas and tightly close with
`stoppers. The tubes can be shaken gently in a shaker at
`room temperature for 15-30min.
`
`3. Separate the denatured protein either by filtration or
`centrifugation. We
`recommend centrifugation where
`denatured proteins can be removed by discarding the
`pellet.
`
`4. Transfer the supernatant
`to another clean and dry
`graduated centrifuge tube and mix with 0.2 volumes of
`0.9% NaCl (to remove non-lipid contaminants). The result(cid:173)
`ing turbid suspension could separate into two phases
`within ca. 2 h. However, a quick centrifugation in any bench
`top centrifuge will separate the two phases within a few
`minutes.
`
`5. Remove the upper layer containing non-lipid contaminants
`carefully with a Pasteur pipette or aspirate off using a suit(cid:173)
`able device.
`
`Par Pharm., Inc.
`Exhibit 1008
`Page 004
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`
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`Extraction
`from
`plant
`tissues
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`Anjni Koul and Rajendra Prasad
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`41
`
`6. Recover the lower clear phase containing most of the lipids
`in another tube and concentrate as described below (for
`complete extraction of membrane lipids, e.g. for the extrac(cid:173)
`lysophospholipids and
`tion of polyphosphoinositides,
`gangliosides special steps are necessary; see Chap. VIII, this
`Vol.). For quantitative extraction, insoluble residue of step 3
`can be re-extracted with fresh solvent.
`
`The method of lipid extraction is essentially similar to animal
`tissues. However, in order to inactivate hydrolytic enzymes,
`e.g. lipases, which are activated in the presence of chloroform
`and diethyl ether, it is necesssary to boil the plant tissue or
`its membrane fraction in propan-2-ol before chloroform:
`methanol extraction (Nichols 1963). In addition, for quantita(cid:173)
`tive extraction of polar lipids, it might be necessary to use high
`salt buffer to partition the solvent phase. A typical protocol for
`the extraction of plant lipids is described below:
`
`1. Boil the plant tissue or protoplast or membrane fraction
`prepared from it in five volumes of propan-2-ol {v/v) for
`5min.
`
`2. Macerise the boiled fraction in a mortar in the presence of
`washed inactive sand and propan-2-ol.
`
`3. Transfer the extract to a glass stoppered tube and add 20
`volumes of chloroform:methanol. Flush the tube with N2
`and shake the mixture for 5 min.
`
`4. Filter the total extract through Whatman # 1 filter disc. For
`quantitative extraction, the residue on the filter paper can
`be re-extracted with fresh solvent mixture.
`
`5. Wash the filtered extract with 0.2 volumes ofNaCl {0.9%) to
`remove non-lipid contaminants and transfer the extract to a
`separating funnel to separate aqueous and non-aqueous
`phases. After flushing with N2, leave the funnel overnight for
`the separation of two phases.
`
`Par Pharm., Inc.
`Exhibit 1008
`Page 005
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`42
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`Extraction of Membrane Lipids
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`6. Collect carefully the lower phase from the separating funnel
`which contains most of the lipids in a stoppered glass
`tube. Flush N 2 and store at 4 °C for evaporation and
`concentration.
`
`This protocol is for the extraction oflipids from yeast cells and
`its plasma membrane fraction. It can also be adopted to other
`membrane preparations of microbial origin.
`1. Mix the harvested yeast cells (500rng dry wt) or plasma
`membrane fraction (15-20mg protein) with methanol
`(lOml) and shake the suspension in a cell disintegrator
`(MSK-Braun Homogeniser) for two periods of 30s at speed
`2 ( 4000 vibrations per min) after addition of 35 g of glass
`beads (0.45-0.5mm diameter).
`2. Add chloroform to the suspension to give the ratio 2: 1 of
`chloroform:methanol (v/v). Stir the suspension for 2h on a
`flat bed stirrer at room temperature.
`3. Filter the suspension using Whatman #1 filter paper. The
`extraction procedure can be repeated on the residue for
`quantitative extraction.
`4. Transfer the extract to a stoppered glass tube and wash with
`0.2 volumes of 0.9% NaCl to remove non-lipid contami(cid:173)
`nants.
`5. Aspirate off the upper aqueous layer that has separated.
`Store the lower layer containing lipids at 4 oc under nitro(cid:173)
`gen atmosphere for evaporation and concentration.
`
`1. Grow the organisms to the desired growth phase in a chemi(cid:173)
`cally defined medium.
`
`2. Harvest the cells by centrifugation at 4000 rpm for 15 min
`and wash the cells three times with distilled water.
`3. Resuspend the cells in propan-2-ol and heat at 70°C for
`45 min, in order to inactivate degradative enzymes such as
`phospholipases (Hitchcock et al. 1986).
`
`Extraction
`from
`yeast
`
`Extraction
`from other
`micro(cid:173)
`organisms
`
`Par Pharm., Inc.
`Exhibit 1008
`Page 006
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`Anjni Koul and Rajendra Prasad
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`43
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`4. Let the suspension cool down to room temperature. Centri(cid:173)
`fuge the suspension to sediment the cells which are recov(cid:173)
`ered after decanting the supernatant.
`
`5. Resuspend the cells in 20 volumes of chloroform: methanol
`(2: 1, v/v) and extract the lipids as follows:
`a) Transfer the cell suspension into a pre-weighed round(cid:173)
`bottomed flask (RBF) and connect a condenser to the
`RBF.
`b) Reflux the suspension in the RBF for 30 min under N 2 gas
`(by connecting to the top of the condenser a balloon full
`of N2 gas), keeping the temperature at 60-70°C and
`avoid boiling.
`c) Filter the solvent mixture using a pre-weighed filter pa(cid:173)
`per, washed previously with chloroform:methanol (2: 1,
`v/v), and retain the filtrate (filtrate 1).
`d) Place the filter paper and the cells back into the pre(cid:173)
`weighed RBF and extract with a fresh solvent mixture of
`chloroform:methanol [2: 1, v/v following the same pro(cid:173)
`cedure as outlined in steps (a) and (b)].
`e) Filter the solvent mixture using another pre-weighed
`filter paper then add the filtrate (filtrate 2) to filtrate 1
`obtained from step (c).
`f) Place the filter paper back into the RBF then dry the RBF,
`filter papers and cells overnight by placing in a desicca(cid:173)
`tor at room temperature.
`g) Weigh the filter paper and cells and subtract from the
`weight of the RBF plus the filter papers. This should give
`the weight of cells.
`h) Evaporate the chloroform:methanol solvent mixture to
`dryness, obtained from the extractions, in a pre-weighed
`RBF using a rotary evaporator (temperature of the water
`bath should be around 70°C).
`i) Place the RBF in a desiccator at room temperature for at
`least 30min and weigh the RBF. The difference will give
`the weight of the crude lipids.
`
`Par Pharm., Inc.
`Exhibit 1008
`Page 007
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`44
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`Extraction of Membrane Lipids
`
`j) The weight of the dry cells will be the weight obtained in
`step (g) plus the weight obtained in step (i).
`
`Evaporation This is a common step applicable to all lipid extracts obtained
`and by following any of the above protocol. Basically, depending
`concentration upon the volume of the extracted lipid, the evaporation can be
`of extracted done as follows:
`lipids
`Small volume. If the extracted lipid volume is small (10-
`15ml), the solvent is best removed from it by evaporation
`under stream ofN2• One or two samples can be concentrated in
`a simple tube where N2 can be bubbled slowly into the solvent
`while the tube is immersed in a water bath set between 65-
`700C.
`In the case of too many samples, use thermostatically con(cid:173)
`trolled heating blocks where N2 can be passed into several
`samples through spaced needles simultaneously (Fig. 1).
`Large volume. If the extracted lipid volume is large, the use
`of rotary evaporator (Fig. 2) for the evaporation of the solvent
`under reduced pressure and temperature is recommended.
`Such apparatuses are commercially available, and are con(cid:173)
`nected to either water or vacuum pump. Using this device, the
`lipid extract can be continuously fed for evaporation and thus
`a large volume of it can be conveniently handled.
`During evaporation, the lipid extract sometimes turns tur(cid:173)
`bid due to the presence of residual water. In such instances,
`one can add 0.1 volume of benzene and continue with the
`evaporation. If necessary, this step can be repeated till a dry
`uniform film of lipid adhering to the walls of rotary flask, is
`obtained.
`
`Purification Further purification of extracted lipids is necessary to analyse
`of them by TLC/GLC/HPLC. The extracted lipids from any source
`extracted can be purified as follows:
`lipids
`
`1. Dissolve
`the crude lipids in
`lOOm! of chloroform:
`methanol (2: 1, v/v) and place in a separatory funnel.
`
`Par Pharm., Inc.
`Exhibit 1008
`Page 008
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`Anjni Koul and Rajendra Prasad
`
`45
`
`Fig. 1. Thermal block for multiple sample concentrator. Such commercially
`available blocks are good if small volumes of lipid extracts are to be concen(cid:173)
`trated. The solvent is evaporated under a stream of N,, which is passed
`through the needles
`
`Par Pharm., Inc.
`Exhibit 1008
`Page 009
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`46
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`Extraction of Membrane Lipids
`
`Fig. 2. Rotary evaporator. It is good for concentrating large volumes oflipid
`extracts under reduced pressure and temperature
`
`2. Add 30 ml of distilled water saturated with chloroform, to
`the separatory funnel. (To saturate the water with chloro(cid:173)
`form, add Sml chloroform to 30ml water).
`
`3. Shake gently in a horizontal manner and carefully
`release the pressure from time to time. Then let the
`separatory funnel stand for at least 10 min. The mixture
`will separate into two layers; the upper layer is an
`aqueous phase containing methanol and impurities, the
`lower layer is an organic phase containing chloroform and
`lipids.
`
`4. Collect the lower phase (chloroform phase) in another
`separatory funnel and repeat steps 2 and 3 once more.
`
`5. Combine the upper phases from the two extractions and
`re-extract with 30 ml chloroform using a separatory funnel.
`
`Par Pharm., Inc.
`Exhibit 1008
`Page 010
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`Anjni Koul and Rajendra Prasad
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`47
`
`6. Combine the two chloroform phases (step 4) and transfer
`into a pre-weighed RBF.
`
`7. Evaporate the chloroform using a rotary evaporator (the
`temperature of the water bath should be around 70 °C).
`
`8. Place the RBF at room temperature for at least 30 min
`in a desiccator containing anhydrous CaC12• Then weigh
`RBF. The difference will give the weight of the pure
`lipids.
`
`9. Dissolve the pure lipids in chloroform ( 10 mg of pure lipids
`should be dissolved in 1 ml chloroform) and transfer into a
`glass vial.
`
`10. Store the glass vial under N2 gas (to prevent possible oxida(cid:173)
`tion of the lipids) at -20°C until needed.
`
`3 Removal of Non-Lipid Contaminants
`
`Lipid extracts have a tendency to trap non-lipid material
`because phospholipids tend to form micelles. The non-lipid
`contaminants are water-soluble substances such as sugars,
`amino groups, urea and salts. Several methods are known
`for the removal of non-lipid contaminants, e.g. removal of
`non-lipids by solvent partitioning, dialysis, evaporation and
`re-extraction, Sephadex chromatography, coprecipitation with
`protein etc.
`
`Most commonly, the non-lipid contaminants present in lipid Removal of
`extract can be removed by simply washing the extract with contaminants
`0.2 volume of water or 0.9% NaCl solution. Calcium and
`magnesium salts should not be used, as they result in low
`recoveries of acidic phospholipids. The technique suffers from
`the disadvantage that it is difficult to apply to large volumes of
`extract, as there is some loss of lipid, particularly that of
`gangliosides, into the upper layer (gangliosides, if needed, can
`be separated from the upper phase by dialysis).
`
`Par Pharm., Inc.
`Exhibit 1008
`Page 011
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`48
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`Extraction of Membrane Lipids
`
`The more elegant and complete, though more time-consum(cid:173)
`ing, method of removing non-lipid contaminations is to carry
`out the washing procedure by liquid/liquid partition chroma(cid:173)
`tography on a column. The method essentially consists of
`passing the lipid extract through a liquid/liquid partition
`column. Sephadex G-25 matrix is used to immobilise the
`aqueous phase which contains non-lipid contaminants and
`gangliosides.
`Removal of non-lipid contaminants by dialysis is not recom(cid:173)
`mended because there is no simple way of knowing when it is
`complete.
`
`4 Storage of Lipids
`
`Standard
`solution
`of
`extracted
`lipids
`
`The extracted dry lipid can be resuspended in known volume
`of chloroform or, if necessary, lipid can be weighed in a
`sensitive balance and a known weight by volume solution
`(e.g. lOmg/ml) in chloroform can be made. The dissolved lipid
`is stored in cold under nitrogen atmosphere, which can be
`maintained by flushing the nitrogen into the container fol(cid:173)
`lowed by tight sealing. This solution is then ready for lipid
`analysis and quantitative estimations of different class of
`lipids.
`
`Storage
`
`Lipids should be stored in glass containers. General con(cid:173)
`tamination can be reduced by not greasing glass tops, by
`using Teflon taps for separatory funnels and columns and by
`sealing desiccators with silicone rubber. Corks and rubber
`stoppers should not be used in lipid work, even polyethy(cid:173)
`lene stoppers can cause contamination and allow solvent to
`escape.
`Since most common lipids contain unsaturated fatty acids,
`care must be taken to avoid auto-oxidation of the sample dur-
`
`Par Pharm., Inc.
`Exhibit 1008
`Page 012
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`Anjni Koul and Rajendra Prasad
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`49
`
`ing storage. Auto-oxidation can be minimised by working with
`oxygen-free solvents and by performing all manipulations
`under nitrogen atmosphere.
`Purified lipid extracts may be stored in tightly closed vials or
`in a container wrapped in aluminium foil at low temperature
`( -20 °C or lower) in the presence of inert solvents and gases, for
`short periods.
`Iflipid solution has to be stored for a longer period, <0.005%
`of antioxidants such as 2,6-di-tert-butyl-p-cresol (or butyl
`hydroxy toluene, BHT), are added to it, which effectively pre(cid:173)
`vents oxidative degradation of unsaturated lipids. The antio(cid:173)
`xidant can be removed by chromatography.
`
`5 Tips, Tricks and Precautions
`
`• During the extraction procedure, contamination from sol(cid:173)
`vents may occur. In order to avoid this, very pure solvents
`(preferably redistilled) should be used.
`• Halide-containing solvents and alcohols have been shown to
`be quite sensitive to light. Hence solvents, in general, should
`be stored in brown bottles.
`• Plastic bottles or tips should not be used during extraction
`procedure. Although they seem to be inert, they contain
`oxidants and low molecular weight polymers, which can
`enter the solvents. Preferably, glass apparatus for the extrac(cid:173)
`tion of lipids and glass pipettes for measuring lipid extracts
`should be used.
`• If highly unsaturated lipids are to be extracted, it is recom(cid:173)
`mended to minimise lipid peroxidation by removing dis(cid:173)
`solved oxygen by slowly flushing nitrogen gas through the
`solvent.
`• The ideal solvent or solvent mixture for extracting lipids
`from tissues should be sufficiently polar to remove all lipids,
`but it should not be so polar that triacylglycerols and other
`
`Par Pharm., Inc.
`Exhibit 1008
`Page 013
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`50
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`Extraction of Membrane Lipids
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`non-polar simple lipids do not dissolve and thus are poorly
`extracted.
`• Care should be taken if diethyl ether is used because of its
`ease of auto-oxidation and the possibility of the presence of
`ether peroxides in the original solvent. Some brands of an(cid:173)
`hydrous diethyl ether contain antioxidants, and normally
`this is indicated on the label; however, BHT found in certain
`brands is not disclosed.
`• Polar lipids are sparingly soluble in hydrocarbon solvents,
`but they dissolve readily in more polar solvents such as
`methanol, ethanol or chloroform. It is seen that the shorter
`the chain length of the fatty acid residue, the greater is the
`solubility of the lipid in more polar solvent.
`• Diethyl ether and chloroform are good solvents for lipids,
`but with this solvent mixture, complex lipids from tissues
`cannot be extracted. Propan-2-ol: hexane mixture has been
`recommended to remove lipids from animal tissue.
`• Many solvents are toxic and even carcinogenic. Care should
`be taken to avoid body contact. If mixture of chloroform and
`methanol spills over a part of body, it causes a severe burn(cid:173)
`ing sensation. The exposed part should immediately be kept
`under cold running water to minimise the burning sensa(cid:173)
`tion, which could last for about lOmin.
`• During extraction of lipids from dry samples, water may be
`added to help in extraction oflipids. Most enzymes are dena(cid:173)
`tured by mixtures containing methanol, but phospholipase
`activity in plants is often enhanced by the presence of ether
`or methanol and loss of complex lipids may result. The en(cid:173)
`zyme may be denatured by blanching the tissues in boiling
`water or hot methanol or by hot propan-2-ol extraction be(cid:173)
`fore treatment with chloroform and methanol.
`
`Table 1 lists common solvents which have been arranged in
`order of increasing dielectric constant (increasing polarity).
`
`Par Pharm., Inc.
`Exhibit 1008
`Page 014
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`Anjni Koul and Rajendra Prasad
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`51
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`Table 1. Common solvents arranged in order of increasing polarity
`
`Solvent
`
`Pentane
`n-Hexane
`Heptane
`Cyclohexane
`Carbon tetrachloride
`Benzene
`Diethylether
`Chloroform
`Ethylacetate
`Pyridine
`Acetone
`Ethanol
`Methanol
`Water
`
`References
`
`Dielectric constant
`
`1.80
`1.89
`1.92
`2.02
`2.02
`2.28
`4.34
`4.80
`6.02
`12.30
`20.70
`24.30
`33.60
`80.37
`
`Folch JLM, Stanley GHS (1957) A simple method for the isolation and puri(cid:173)
`fication of total lipids from animal tissues. J Bioi Chern 226:497-509
`Hitchcock CA, Barrett-Bee KJ, Russell NJ (1986) The lipid composition of
`azote-resistant strains of Candida albicans. J Gen Microbiol 132:2421-
`2431
`Nichols BW (1963) Separation of the lipids of photosynthetic tissues:
`improvement in analysis by thin layer chromatography. Biochim
`Biophys Acta 70:417-422
`
`Par Pharm., Inc.
`Exhibit 1008
`Page 015