- Email: [email protected]
[57] Preparation and analysis of phosphatides
328
LIPIDS AND STEROIDS
[57]
Titration. After cooling to 20 °, pipet a 4-ml. aliquot of the benzene extract into a 5-ml. test tube, and evaporate the benzene to dryness in a tube heater or on a steam bath under a stream of nitrogen. Add exactly 1 ml. of alcoholic thymol blue, taking care to wash down the walls of the tube. By means of a fine capillary, bubble a fine stream of nitrogen or C02-free air through the solution for several minutes in order to remove any carbon dioxide. Then titrate the fatty acids with tetramethyl ammonium hydroxide from a micrometer buret whose tip extends below the surface of the liquid. The end point is reached when the indicator changes from a bright yellow to a persistent yellowish-green tinge. Place a stoppered control tube containing 1 ml. of the acid alcoholic thymol blue in permanence next to the solution being titrated. A reagent blank is run with each set of determinations, the titration value being about 1 #l. Calculations. V = net titration value in microliters; N = normality of N(CHs)4OH; and W = body fluid in milligrams or microliters.
Meq. of fatty acid in aliquot titrated -
V×N
1000
(l)
F a t t y acids in body fluids, usually expressed as milliequivalents per liter, are calculated as follows: V > ( N >(1000 5 Meq./1. = W × ~ (2) Normal plasma values vary from 6 to 20 meq./1. To convert to milligrams of fatty acids per 100 ml., multiply the milliequivalents per liter by 27.72, the average molecular weight of blood fatty acids, according to Stoddard and Drury.**
[57] Preparation and Analysis of Phosphatides 1 By ~/~ARJORIE B. LEES
Assay Methods The quantitative analysis for total and individual phosphatides presents numerous problems associated with the completeness of their extraction, the removal of nonlipid contaminants, hydrolysis procedures, and the specificity of the methods. The complete extraction of the phos1The excellent books by H. Witcoff, "The Phosphatides" (Reinhold Publishing Corp., New York, 1951) and H. J. Deuel, Jr., "The Lipids" Volume I (Interscience Publishers, Inc., New York, 1951) are recommended for further references and discussion of much of the material presented.
[57]
PHOSPHATIDES
329
phatides and the removal of nonlipid contaminants are prerequisites for any method for the determination of total phosphatides (Vol. III [55]). The most commonly used method is the analysis of an adequately extracted, dialyzed sample for phosphorus. This is as accurate as and much simpler than the oxidative method of Blooff or the determination of fatty acids recommended by Artom. 3 Analyses for lipid phosphorus can be carried out by the method of Sperry 4 or by any other suitable procedure. The phosphorus value obtained is converted to phosphatides by means of a factor based on the average phosphorus content of the phosphatides. Although various investigators have used experimentally determined factors, multiplying the lipid P by 25 (based on an average P content of 4%) is adequate for most purposes. The best methods for the analysis of individual phosphatides are those based on a study of their hydrolysis products. Methods dependent on the solubility of the lipids are completely inadequate, since the solubility of the individual lipids is influenced by the presence of other lipids in a mixture. The cephalin fraction, for example, is insoluble in alcohol, whereas the phosphatidyl ethanolamine isolated therefrom is completely alcohol-sohible. After hydrolysis of the phosphatides, a combination of several analytical techniques can be used to determine the amounts of the main components of the mixture. Table I shows the distribution of various chemical components among the principal phosphatides. Phosphatides such as cardiolipin, acetal phosphatides, and phosphatidic acids have been omitted from this discussion, since they either have been isolated only from special tissues or have not been sufficiently characterized to warrant their inclusion here. Brief descriptions of their preparation may be found in Witcoff's book.' The following relationships can be seen from Table I: (1) lecithin and sphingomyelin are the only known choline-containing lipids; (2) the amount of the lecithin plus cephalin fraction is represented by the amount of glycerol present; (3) the total amino nitrogen value gives the sum of the phosphatidyl ethanolamine and phosphatidyl serine fractions. Glycerol is determined by the method of Blix; 5 inositol can be determined microbiologically or chemically;6 methods for the quantitative determination of choline, ethanolamine, serine, and sphingosine are described in Vol. III [59]. In evaluating the analytical results, however, the effect of the minor phosphatides should not be overlooked. 2 W. R. Bloor, J. Biol. Chem. 82, 273 (1929). C. Artom, Bull. soc. chim. biol. 14, 1386 (1932). 4 W. 5/[. Sperry, Ind. Eng. Chem. Anal. Ed. 80, 46 i1938). 5 G. Blix, Mikrochim. Acta 1, 75 (1937). 6 D. W. WooIley, J. Biol. Chem. 140, 453 (1941) ; B. S. Platt and G. E. Glock, Biochem, J. 37, 709 (1943).
330
LIPIDS AND STEROIDS
[57]
Methods based on preferential hydrolysis constitute a slightly different approach to the analysis of individual phosphatides. Schmidt et al. 7 have shown t h a t the P of monoaminophosphatides becomes acid-soluble under conditions where sphingomyelin remains intact. This has been used as the basis for the assay of sphingomyelin described below. Hack s has developed a similar method for blood phosphatides based on the fact t h a t the choline from lecithin can be hydrolyzed whereas t h a t from sphingomyelin remains intact. After acidification and removal of the unreacted TABLE I CHEMICAL COMPOSITION OF THE PRINCIPAL PHOSPHATIDES
P N a-NH~-N ~ NH2-N ~ Choline [ Glycerol Lipid Atom grams Phosphatidyl choline (lecithin) Cephalins Phosphatidyl ethanolamine Phosphatidyl serine Diphosphoinositide Sphingomyelin
Moles
1
1
0
0
1 1 2 1
1 1 0 2
0 1 0 0
1 1 0 0
By the ninhydrin-CO2 method of D. D. Van Slyke, R. T. Dillon, D. A. MacFadyen, and P. Hamilton, J. Biol. Chem. 141, 627 (1941). b By the nitrous acid manometric method of D. D. Van Slyke, J. Biol. Chem. 83, 425 (1929). sphingomyelin, the filtrate is analyzed for P and choline. F r o m these values and the total P, values for lecithin, cephalin, and sphingomyelin can be calculated. I t should be noted t h a t these hydrolysis procedures are not necessarily applicable to all tissues. Brain lipids in particular often behave differently from those of other tissues. For example, Sperry and Brand's hydrolysis procedure for sphingomyelin could not be applied satisfactorily to brain lipids2 Criteria f o r P u r i t y of Phosphatides. The development of adequate criteria for the determination of the degree of purity of phosphatide preparations still awaits future research. Recent studies using countercurrent distribution and chromatographic techniques have made a beginning in t h a t direction. It is known t h a t the individual phosphatides isolated thus far are not single chemical compounds but are mixtures of closely related substances differing at least in their f a t t y acid residues. 7 G. Schmidt, J. Benotti, B. Hershman, and S. J. Thannhauser, J. Biol. Chem. 166, 505 (1946). 8 M. H. Hack, J. Biol. Chem. 169~ 137 (1947). J. Folch and W. M. Sperry, Ann. Rev. Biochem. 17, 147 (1948).
[57]
PHOSPHATIDES
331
It is significant that, as yet, no synthetic phosphatide has been prepared which is identical with the substances isolated from tissues. Since we are not dealing with a homogeneous chemical compound, the determination of physical constants is not sufficiently precise to be of value in a consideration of the purity of a phosphatide sample. Melting point and solubility, for example, are affected by the chain length and degree of unsaturation of the fatty acid residues. At the present state of knowledge the best approach to a consideration of the degree of purity of a phosphatide preparation is by means of chemical analyses for the amounts of impurities present. The following points should be noted: (1) The complete removal of nonlipid contaminants should be ensured by adequate dialysis of the preparation. It is known that lipids can carry impurities with them through lengthy fractionation procedures. (2) The elemental chemical analysis and the amounts of the hydrolysis products obtained should be consistent with the accepted formula for that phosphatide. (3) No hydrolysis products other than those required by the accepted formula should be present; phosphatide samples should be free of carbohydrates and cholesterol. (4) Hydrolysis products should be present in large enough amounts to exclude the possibility of their being contaminants. Proof that a constituent is combined chemically can best be obtained by a study of the kinetics of its liberation by several agents. (5) the N : P ratio, although widely used in the literature, is not a particularly sensitive criterion for purity, since large amounts of contaminants may be present without affecting the ratio significantly. (6) All the nitrogen (or phosphorus) should exist in the form required by the accepted formula for that phosphatide. With the exception of sphingomyelin, which contains both sphingosine and choline, the nitrogen should be present as a single chemical constituent. Some of the above points will be discussed under the individual phosphatides.
Preparation of Lecithin (Phosphatidyl Choline) Most of the methods for the preparation of lecithin depend on the precipitation of its cadmium salt from an alcoholic solution. The method of Pangborn, 1° described below for the preparation of egg lecithin, is a simplification of that of Levene and his co-workers. 11,12 In Pangborn's method the lecithin-CdCl~ complex is broken with a specific combination of organic solvents, after which the cadmium is removed with dilute alcohol (Fig. 1). l0 M. Pangborn, J. Biol. Chem. 188, 471 (1950). ~1 p. A. Levene and H. S. Simms, J. Biol. Chem. 48, 185 (1921). ~2 p. A. Levene and I. P. Rolf, J. Biol. Chem. 72, 587 (1927).
332
LIPIDS A N D
STEROIDS
[57]
Step 1. Extraction and Preparation of Lecithin-CdCl2. T h e yolks of twelve fresh eggs are stirred for a few seconds in a Waring blendor; 200 ml. of acetone is added, and the mixture is stirred for about 30 seconds, transferred to a beaker, stirred thoroughly with 400 ml. more of acetone, and Egg yolks ÷acetone
Soluble
Insoluble ÷95% ethanol
Soluble
Insoluble
50% CdCI:,
Soluble
Insoluble +CHCls:C2H~0H. 1:7, 3 times
Insoluble
Soluble
l
-i-Pelroleura ether:SO% alcohol, 3:10
Lower phase ffi 80% alcohol
Upper phase petroleum ether
I
Re-extract
with 80% alcohol 2 times
7
~oluble
_5
Insoluble /
)~-I-CHCIs:30 % C~HbOH, 1: 1
phase ffi C~H~OH
Lower phase ffi CHCI3
Upper
!
)Dried~-t-et her: acetone, 5:1, 4°
Insoluble
Soluble Lecithin
Fro. I. filtered by suction. T h e filter cake is extracted five times in the blendor with 200-ml. portions of acetone. T h e creamy white yolk powder thus obtained is extracted for 30 minutes in a mechanical shaker with 800 ml. of 95% ethanol and filtered by suction. This alcoholic extract is precipitated with a slight excess of 50 % aqueous CdC12 (approximately 15 ml.). After standing for 1 hour at 4 °, the mixture is filtered by suction and the precipitate is washed twice with acetone while still on the filter.
[~7]
PHOSPHATIDES
333
Step 2. Purification of Lecithin-CdCl~. The precipitate is dissolved in 100 ml. of chloroform. The slightly cloudy, faintly brown solution is poured with constant mixing into 700 ml. of ethanol containing 10 ml. of 50% aqueous CdC12 to prevent dissociation of the lecithin salt. After intermittent shaking for 10 minutes at room temperature, the mixture is filtered by suction. The precipitate is redissolved in chloroform and treated with ethanol and CdC12 as before, except that it is allowed to stand for 30 minutes with frequent shaking. A third chloroform-alcohol precipitation is carried out as above. The cadmium salt is then suspended in 150 ml. of petroleum ether, and to it is added 500 ml. of 80% alcohol previously saturated with petroleum ether and containing 0.1% CdCl~. After vigorous shaking in a separatory funnel, the alcohol layer is drawn off and the petroleum ether layer is re-extracted twice with a total of 100 ml. of the above 80% alcohol mixture per gram of material remaining in the petroleum ether layer (as determined by drying an aliquot). The combined alcohol extracts are concentrated to two-thirds the original volume to remove the petroleum ether. The concentrated lecithin-CdCl~ mixture is left at about - 5 ° overnight, after which it is filtered by suction. Step 3. Removal of Cadmium. The lecithin-CdC12 precipitate is dissolved in 150 ml. of chloroform, an equal volume of 30% ethanol is added, and the mixture is shaken vigorously for 5 minutes. Under these conditions the lecithin double salt dissociates, and the CdCb. is washed out in the dilute alcohol. Four extractions with 30% alcohol should suffice to remove all the cadmium. An aliquot of the aqueous-alcoholic layer may be tested for the presence of cadmium ions with a drop of 5% AgN03. One extra extraction should be carried out to ensure complete removal of the cadmium. Step ~. Final Purification. The chloroform solution is dried by vacuum distillation of the solvent. The lecithin is dissolved in 100 ml. of anhydrous ether, and to it is added 20 ml. of acetone. The mixture is placed at 4 ° overnight, and the finely flocculent precipitate which forms is removed by filtration over a Btichner funnel. The clear filtrate is evaporated to dryness in vacuo, and the purified lecithin is dissolved in absolute alcohol. Yield, 7 grams; P, 4.03%; N, 1.98%; NH2-N, 0.02%; N : P , 1.06:1. Modifications for Tissues Hanahan and Jayko TM have isolated an individual, completely unsaturated lecithin from baker's yeast by a method involving a final purification of the material on an aluminum oxide column. Since their 1~ D. J. Hanahan and M. E. Jayko, J. Am. Chem. Soc. 74, 5070 (1952).
334
LIPIDS AND STEROIDS
[57]
preparation has been well characterized, it should prove particularly useful for enzyme studies. The preparation of lecithin from tissue extracts is more difficult, and each material presents special problems. A summary of Pangborn's modification for the preparation of beef heart lecithin is as follows. Step 1. Removal of Cardiolipin. 13 6.2 kg. of fresh, defatted beef hearts is ground in a meat grinder. The minced tissue is extracted twice at room temperature with 1.2 ml. of acetone per gram of fresh tissue. The acetone is removed by suction; the residue from the second extraction is dried before a fan until the acetone odor is gone. It is ground to a fine powder which is then extracted three times with 2 1. of 95 % methanol for each 300 g. of powder, each extraction being carried out for a week at room temperature with frequent shaking. After the final extraction, the residue is washed with methanol and discarded. As each extract is collected, it is precipitated with 20% aqueous BaC12 and stored at 3 to 6 °. The pooled extracts are centrifuged; the precipitate can be processed for cardiolipin while lecithin is prepared from the supernatant fluid. Step 2. Precipitation of Lecithin-CdCl2 and Removal of Cadmium. The procedure is then essentially as described above. Lecithin is precipitated from its alcoholic solution by the addition of excess 50% aqueous CdC12; the precipitate is taken up in 200 ml. of chloroform and precipitated with 1400 ml. of ethanol a total of three times, after which the cadmium is removed. The cadmium-free chloroform solution is evaporated to dryness and taken up in 180 ml. of anhydrous ether. To the slightly cloudy solution is added 36 ml. of acetone. The precipitate which forms when the mixture is refrigerated overnight is removed by filtration, and the clear filtrate is evaporated to dryness and redissolved in a small amount of alcohol. Yield, 18 g.; NH2-N, 0.25%. Step 3. Further Purification. The alcoholic solution is made alkaline to phenolphthalein with 10 ml. of saturated aqueous Ba(OH)2, the mixture is immediately neutralized with CO2, and 2 ml. of saturated NaCl is added with vigorous shaking to flocculate the precipitate. The precipitate is removed by gravity filtration, and the clear filtrate is reprecipitated with CdC12. The cadmium salt is precipitated twice by pouring its chloroform solution into alcohol and then fractionated once by the petroleum ether-80% alcohol method described for egg lecithin. The cadmium is then removed, and the lecithin is dissolved in ether to which 20 % of its volume of acetone is added. After refrigeration at - 5 ° overnight, the small amount of precipitate is removed by filtration, and the filtrate is dried and dissolved in a small amount of alcohol. Yield, 10.5 g.; P, 4.21%; N, 1.94%; N : P , 1.01; NH2-N, 0.01%; iodine number, 83.7. ~s M. Pangborn, J. Biol. Chem. 161, 71 (1945).
[57]
PHOSPHATIDES
335
Step 4. Purification for Serological Tests. 1~ The above preparation can be separated chromatographically into fractions of different serological activity (in the Wasserman test for syphilis). A glass column (50 X 250 ram.) is packed with Magnesol-Celite. After impregnation of the column with benzene, 500 ml. of lecithin in 15 ml. of benzene is added at the top of the column, which is then developed with 500 ml. of 2% tert-butyl alcohol in benzene. The column is extruded, and a narrow vertical band is streaked with 1% potassium permanganate in 2.5 N NaOH. The zones which thus show are then eluted with absolute ethanol. This procedure can be applied successfully to commercial lecithin preparations or to lecithin-CdCl~ complexes. Residual impurities can also be removed with aluminum oxide columns. 15
Properties Pure lecithin is a whitish, paraffinlike substance which is extremely hygroscopic. When dry it can be ground to a powder, but on taking up water it becomes a waxy, sticky mass. On exposure to light and air the material darkens and develops a disagreeable odor owing to the instability of the fatty acids. No definite melting point can be determined. Lecithin is readily soluble in methanol, ethanol, benzene, ether, petroleum ether, chloroform, carbon tetrachloride, and carbon disulfide. It is also soluble in pyridine, glycerol, and acetic acid. It is, however, insoluble in methyl acetate and acetone. Lecithin may be precipitated from its water emulsion by the addition of acetone, acids, or inorganic salts. Lecithin containing completely saturated fatty acids (hydrolecithin) is insoluble in ether and is found as a contaminant in the sphingomyelin fraction unless special precautions are taken to remove it. Lecithin is easily hydrolyzed by either acid or alkali. Hack, s using normal KOH at 37 ° for 16 hours, found glycerophosphate, choline, and fatty acids among the hydrolysis products. Since migration of the phosphoric acid radical occurs during hydrolysis, the configuration of the naturally occurring substance cannot be determined from the configuration of the isolated glycerophosphoric acid. 16 The best evidence is that most, if not all, of the lecithin occurs naturally in the a configuration. 17 Lecithin probably exists in the form of a zwitterion. Some sort of internal neutralization is to be expected because of the presence of a strongly acidic and a strongly basic group. Lecithin in solution is essentially neutral and has almost no buffering power in the physiological range. The 14F. A. H. Rice and A. G. Osler, J. Biol. Chem. 189, 115 (1951). 15M. Faure and J. Legault-Demare, Bull. soc. chim. biol. 82, 509 (1950). 16j. Folch, J. Biol. Chem. 146, 31 (1942). 17E. Baer and M. Kates, J. Biol. Chem. 17li~79 (1948).
336
LIPIDS AND STEROID)~
[57]
isoelectric point is rather difficult to determine, but the best values place it in the neighborhood of 6.418 (theory = 7.5). 19 P u r i t y . The most sensitive criterion for the purity of a lecithin preparation is the absence of a-amino nitrogen. 2° No base other than choline should be present. Lecithin preparations should have a N: P ratio of 1 : 1; all the phosphorus should be saponifiable under the conditions of the procedure of Schmidt et al.; and theoretical amounts of choline should be obtained after adequate hydrolysis.
Preparation of Cephalin Phosphatides The multiple nature of the cephalin fraction was first recognized by Folch and Schneider. 21 In the method of Folch 22 described below, the crude cephalin mixture from brain is separated into three different fractions on the basis of their differing solubilities in chloroform-ethanol mixtures. Increasing amounts of ethanol are added to a chloroform solution of cephalin, and the precipitates are collected at various concentrations of ethanol. In this manner three fractions can be isolated: (1) diphosphoinositide, which shows relatively low solubility in alcohol; (2) phosphatidyl serine; and (3) phosphatidyl ethanolamine, which is freely soluble in alcohol. The success of the separation of the crude cephalin mixture is dependent on the fact that phosphatidyl ethanolamine is present as the free ampholyte whereas the other phosphatides are in the salt form. In previous procedures the use of HC1 resulted in their conversion to the free acid form which is evidently more difficult to separate. Aside from the preparation described below, probably the only pure phosphatidyl ethanolamine preparation has been that of Rudy and Page. 2a Although analysis shows the presence of phosphatidyl serine in various tissues, it has never been prepared in a pure state from any tissue except brain. Inositol phosphatides have been reported from a variety of sources. Woolley24 isolated an inositol-containing phosphatide from soybeans which he named lipositol. No formula has been proposed for this compound, although galactose, ethanolamine, tartaric acid, saturated and unsaturated fatty acids, phosphoric acid, and inositol have been identified among its hydrolysis products. It is possible that lipositol is a mixture of closely related lipids. ~sH. B. Bull and V. L. Frampton, J. Am. Chem. Soc. 58, 594 (1936). 19H. Fischgoldand E. Chain, Biochem. J. 28, 2044 (1934). 20D. D. Van Slyke, R. T. Dillon, D. A. MacFadyen, and P. Hamilton, J. Biol. Chem. 141, 627 (1941). ~1j. Folch and H. A. Schneider, J. Biol. Chem. 187, 51 (1941). ~ J. Folch, J. Biol. Chem. 146, 35 (1942); 177, 497 (1949). 2s H. Rudy and I. H. Page, Z. physiol. Chem. 198, 251 (1930). ~ D. W. Woolley,J. Biol. Chem. 147, 581 (1943).
[57]
PHOSPHATIDES
337
Step 1. Preparation of Crude Brain Cephalin. Fresh ox brains are freed of their membranes, and 100-g. portions of tissue are ground in a Waring blendor with 300 ml. of acetone for 2 minutes. The portions are combined, and acetone is added until there is at least 3.8 ml./g, of tissue. After a few minutes the acetone is removed by suction and discarded. The precipitate is extracted once more with acetone, once with alcohol, and twice with petroleum ether, 4 ml. of each solvent being used per gram of original tissue. The two petroleum ether extracts are combined and dried completely by vacuum distillation. This dried material is then suspended in 200 ml. of ethyl ether for each kilogram of original tissue and allowed to stand in the refrigerator for a day or two. If a clear supernatant does not separate within three days, water is added to the extent of 1% of the volume of ether. If this does not result in the appearance of a clear upper fraction, the ether suspension should be diluted one-fold with ether. Once a supernatant solution amounting to about one-fourth of the total volume is obtained, the suspension is centrifuged and the precipitate is washed twice with cold ether. The combined ether extracts are dried by vacuum distillation at room temperature, and the residue is dissolved in 50 ml. of ether for each kilogram of initial brain tissue. The ethereal solution is placed in the refrigerator overnight, and any precipitate which forms is discarded. The solution is diluted with an equal volume of ether, and 5 vol. of alcohol is added with stirring to precipitate the cephalin. After the mixture stands at room temperature for about 1 hour, a clear supernatant is formed. The precipitate is collected on a Biichner filter and suspended in 100 ml. of acetone per kilogram of original brain tissue. The suspension is shaken for 40 minutes to remove acetone-soluble impurities. After removal of the supernatant fluid by suction, acetone is again added to the precipitate and the procedure is repeated. Yield, 15 g. of a tan cephalin powder per kilogram of initial tissue; C, 55.2 %; P, 4.13 %; N, 1.59 %; NH~-N, 1.51%; a-NH~-N, 0.76%; NH2-N:N, 0.96; P : N , 1.17. Step 2. Fractionation of Brain Cephalin by Chloroform-Alcohol Method. One gram of the crude cephalin preparation described above is dissolved in 8 ml. of chloroform, and to it is added 1.45 times as much alcohol as chloroform by volume. A turbidity develops and, after standing for about 1 hour, the.-~ixture is centrifuged and resolves itself into a viscous underlayer (fractions I and II, inositol phosphatide fraction) and a clear supernatant. The supernatant is decanted, and to it is added 25 ml. of alcohol. The precipitate (fraction III, phosphatidyl serine) which appears is collected on a Bfichner filter and dried. The filtrate is concentrated in vacuo to half its volume and allowed to stand in the refrigerator for 2 or 3 days. The precipitate (fraction IV) is removed by filtration in the
338
LIPIDS AND STEROID
[57]
cold and dried. The filtrate is concentrated to 1 ml., and to it is added 5 ml. of acetone. After standing for 1 d a y in the refrigerator, the acetoneinsoluble precipitate (fraction V, phosphatidyl ethanolamine) is removed by filtration and dried. T a b l e I I gives an analysis of the above fractions. TABLE II ~ ANALYSIS OF FRACTIONS ISOLATED FROM BRAIN CEPHALIN BY CHLOROFORMALCOHOL METHOD
Components C P N Amino N b ~-Amino N ¢ Inositol Iodine No. Ash Yield, g. per 100 g. eephalin
Fraction Fraction Fraction Fraction V I II III (phospha(inositol (inositol (phosphatidyl phospha- phospha- tidyl Fraction ethanoltide), tide), serine), IV, amine), % % % % % 55.0 4.25 1.15 1.15 0.70 6.8 65.0 16.7 22.0
59.0 3.86 1.36 1.36 0.80 3.4 10.0
60.2 3.58 1.62 1.64 1.47 <0.20 39. g 12.8 27.0
63.0 3.60 1.75 1.60 0.60 <0.20 8.0
66.1 3.65 1.78 1.50 < 0.02 <0.20 78.0 2.5 15.0
From J. Folch, J. Biol. Chem. 146, 35 (1942). b By the nitrous acid manometric method of D. D. Van Slyke, J. Biol. Chem. 63, 425 (1929). c By the ninhydrin-CO~ method of D. D. Van Slyke, R. T. Dillon, D. A. MacFadyen, and P. Hamilton, J. Biol. Chem. 141, 627 (1941).
Step 3. Purification of Inositol Phosphatide by Chloroform-Methanol Method. T h e viscous underlayer (fractions I and I I ) is t r e a t e d with ethanol, and the solid precipitate which forms is collected on a Biichner filter and dried. I t is then processed at 4 ° as follows: One g r a m of crude inositol phosphatide is dissolved in 12 ml. of chloroform, and to the clear solution is added 22 ml. of methanol. T h e mixture is shaken for 30 minutes in a shaking machine, after which the precipitate is collected by centrifugation. I t is then redissolved in 12 ml. of chloroform, 22 ml. of m e t h a n o l is added to the solution, and the procedure is repeated as before as long as the s u p e r n a t a n t solution contains material with less t h a n 4.5% P. I f at a n y point the precipitate fails to dissolve completely in chloroform, the insoluble material can be removed by precipitation with 2 col. of ether. I f the material has not been previously dialyzed, twelve
[57]
PHOSPHATIDES
339
successive precipitations usually suffice, but as m a n y as thirty precipitations m a y be required on a dialyzed preparation. The crude diphosphinositide so obtained contains large amounts of water-soluble contaminants. These m a y be removed by dialysis as described below. Table I I I gives the analysis of the fractions obtained before and after dialysis. TABLE III ANALYSIS OF BRAIN DIPHOSPHOINOSITIDE a
Component
Before dialysis, %
Mter dialysis, %
31.5 0.79 11.08 6.10 0.10
46.0 0.4-0.6 7.0-7.3 <0.05 <0.02 0.1-0.4 0.4-1.8 9.0 2 i. 0 { <0.01 0.64 2.88 0.5-0.6
C N P Inorganic P a-NH2-N NH2-N (after acid hydrolysis) Carbohydrate (as galactose) Glycerol Inositol K Na Ca Mg Yield, g./kg, fresh tissue
1.7 4.9 8.05 3.25 0.30 1.30 1.0
a Data from J. Folch, J. Biol. Chem. 177, 505 (1949). Step 4. Dialysis of Fractions. A 3 % aqueous emulsion of each of the fractions is prepared by shaking 1 g. of material with 30 ml. of water until homogeneous. The emulsion is dialyzed at 4 ° for 4 days against distilled water with several changes of the outside liquid. The undialyzable fraction is then lyophilized, and the fluffy white powder which is obtained is suspended in acetone, collected on a Biichner filter, and dried. Step 5. Purification of Phosphatidyl Serine. 25 The above method results in phosphatidyl serine preparations which are 85 to 90% pure; i.e., between 85 and 90% of the total nitrogen is present as a-amino nitrogen. If lower values are obtained or if slightly greater purity is required, the following procedure is recommended: One gram of the preparation is dissolved in 10 ml. of chloroform, and to it is added 16.5 ml. of absolute ethanol. A turbidity develops which, on standing or by centrifugation, resolves itself into a viscous underlayer and a clear supernatant solution. The supernatant solution is decanted, and to it is added 30 ml. of absolute ~5j. Folch, J. Biol. Chem. 174, 439 (1948).
340
LIPIDS AND STEROIDS
[57]
ethanol. The precipitate which separates is collected and dried. It contains a higher concentration of a-amino nitrogen than does the mother substance. Phosphatidyl serine preparations of greater than 92% purity are not obtained consistently. The main contaminant appears to be phosphatidyl ethanolamine, since all the nitrogen can be accounted for as amino nitrogen. 28 It is essentially free of cerebrosides ( < 0 . 1 % carbohydrate), lecithin and sphingomyelin ( < 0 . 1 % choline), and cholesterol (<0.1%).
Properties of Phosphatidyl Ethanolamine Freshly prepared phosphatidyl ethanolamine is a slightly sticky white powder which becomes dark brown and stickier on standing in the dark in a vacuum desiccator. This change in physical appearance is evidently not accompanied by any observable change in elementary composition. The material contains 1.7% water, which can be removed at 80 ° in vacuo. However, it returns to its original weight on storage in a desiccator over CaC12. As isolated from brain by neutral solvents, it is ash-free. Phosphatidyl ethanolamine, unlike the cephalin mixture from which it is derived, is freely soluble in alcohol. It is also soluble in chloroform, petroleum ether, carbon disulfide, benzene, hot acetic acid, and wet ether. It is insoluble in anhydrous ether and acetone and ~orms an emulsion with water. It is hydrolyzed relatively easily by either acids or alkali. The iodine number of the product isolated by Folch is 78, indicating the presence of two double bonds for each P atom. The few physical measurements available have been carried out on impure preparations and therefore do not warrant discussion. Purity. All the nitrogen of phosphatidyl ethanolamine should be present as amino nitrogen as determined by the nitrous acid procedure of Van Slyke. 26 The presence of serine can be excluded by the absence of a-amino nitrogen. A phosphatidyl ethanolamine preparation should be free of choline and inositol; all the phosphorus should be saponifiable under the conditions of the procedure of Schmidt et a l . f and it should show a N : P ratio of 1:1.
Properties of Phosphatidyl Serine Phosphatidyl serine is a free-flowing white powder with a slight tendency to darken. After long periods of storage in vacuo in the dark at room temperature there is no change in its elementary composition but there is some sort of molecular rearrangement involving a sharp drop of its a-amino nitrogen and primary amino nitrogen content. Storage in chloroform solution in a dry icebox seems to be more satisfactory. Phosphatidyl 26 D. D. Van Slyke, J. Biol. Chem. 83, 425 (1929).
[57]
PHOSPHATIDES
341
serine, like phosphatidyl ethanolamine, contains a small percentage of water. It is freely soluble in chloroform, ethyl ether, and petroleum ether, but insoluble in ethanol, methanol, and acetone. In the presence of base, phosphatidyl serine forms a stable, concentrated emulsion with water. However, acidification of the emulsion to pH 1.5 results in a complete precipitation of base-free phosphatidyl serine. The process may be reversed by the addition of theoretical amounts of alkali. Phosphatidyl serine is a strongly acidic compound, having one basic group (the - - N H 2 group of serine) and two acidic, groups (the - - C O O H group of serine and one group from phosphoric acid). Therefore, at physiological pH it binds one equivalent of base for each atom of P. This is shown by the fact that phosphatidyl serine isolated from brain by the use of neutral solvents and freed of water-soluble impurities by dialysis contains Na and K. Preparations obtained by the method described above contain much more K than Na, but Ca and Mg are absent. The same preparations also give theoretical values for an equimolar mixture of oleic and stearic acids. Glycerophosphoric acids, L-serine, and fatty acids have been isolated as hydrolysis products of phosphatidyl serine in approximately 1:1:2 proportions. Intact phosphatidyl serine reacts with ninhydrin and with nitrous acid in the same way as does an a-amino acid, showing that both the carboxyl and the amino groups are free. Its inability to react with periodic acid indicates that the hydroxyl group must be combined. Purity. All the nitrogen of a phosphatidyl serine preparation should be present as free a-amino nitrogen; the presence of any other type of nitrogen indicates the existence of impurities. This is the only quantitative method for differentiating phosphatidyl serine .from phosphatidyl ethanolamine, as other criteria listed under phosphatidyl ethanolamine apply to both substances.
Properties of Diphosphoinositide Diphosphoinositide prepared from brain by neutral solvents is an acidic phosphatide which is obtained as a salt of calcium and magnesium. ~7 It is a white, gritty powder which is not emulsified in water and is insoluble in most organic solvents except wet chloroform. It can be converted to the potassium salt which forms a stable emulsion with water but is insoluble in the usual organic solvents. It is possible to obtain a solution of this material in an organic solvent by diluting a 5 % emulsion with methanol or 2:1 chloroform-methanol. The base can be removed by treatment with dilute acid, indicating that the bases are present in a saltlike linkage. The base-free diphosphoinositide is readily soluble in 27j. Folch, J. Biol. Chem. 177, 505 (1949).
342
LIPIDS AND STEROIDS
[57]
water, forming an acid solution which, on titration, uses one equivalent of base per atom of P present. Brain diphosphoinositide is both alkali- and acid-labile. Among the products of hydrolysis, fatty acids, glycerol, and inositol metadiphosphate have been isolated in approximately equimolar proportions. The chemical structure of inositol metadiphosphate has been established by Folch by (1) elementary analysis, (2) isolation of the theoretical amount of inositol, (3) titration to pH 8.7, which shows the presence of two free acid groups for each phosphoryl radical, and (4) a study of the reaztion products with HI04, which shows that the two phosphoryl groups are in the meta position on the inositol. The attachment of the remaining constituents in the molecule is as yet unknown. The nitrogen and carbohydrate are probably present as impurities. The following formula has been postulated on the basis of the experimental evidence, where R and R' stand for unknown radicals:
j0R
HC-0.P0
J\\o
ItCOH
HCOH OR'
HCOH
\J
HC.0.P0
HCOH Purity. Diphosphoinositide preparations should be free of nitrogen and choline; phosphorus and inositol should be present in a 2:1 ratio.
Sphingomyelin (Monoacylsphingosylphosphoryl Choline) Assay Method Principle. The older methods for the determination of sphingomyelin depended either on its solubility, ~8 which gave low results, or on its precipitation as a reineckate, ~9 which gave high results. The analysis for sphingomyelin developed by Schmidt et al. 7 is based on the fact that these compounds are much more resistant to alkali than are the glycerophosphatides. The conditions of the procedure are such that the selective saponification of the glycerophosphatides is brought about. Since sphingomyelin is represented by the difference between the total and the saponi2s E. Kirk, J. Biol. Chem. 123, 623 (1938). 29 B. N. Erickson, I. Avrin, D. M. Teague, and H, H. Williams, J. Biol. Chem. 136, 671 (1940).
[57]
PHOSPHATIDES
343
fiable phosphorus, the values can only be considered approximate in cases where the sphingomyelin content of the tissue is small. On the other hand, it is very useful for detecting the presence of small amounts of hydrolecithin which often contaminates sphingomyelin samples. Procedure. Suitable aliquots of the lipid samples to be tested are placed in wide Pyrex test tubes and evaporated to dryness on a water bath. Each sample is emulsified with 5 ml. of N potassium hydroxide at 37 ° for 24 hours. Schmidt recommends continual shaking of the emulsion, but we have not found this to be necessary. The emulsion is precipitated by the addition of 1 ml. of 5 N hydrochloric acid and 5 ml. of 10% trichloroacetic acid. After standing for 1 hour at room temperature (to hydrolyze acetal phosphatides), the samples are centrifuged and filtered. The phosphorus in the original lipid solution and in the clear trichloroacetic acid filtrate is determined. The difference between the two values represents the nonsaponifiable or sphingomyelin phosphorus.
Preparation Sphingomyelin prepared by the usual methods 3°,31 is contaminated with a large proportion of hydrolecithin. The observation that sphingomyelin is resistant to N alkali at 37 °, in contrast to hydrolecithin which is completely hydrolyzed under those conditions (see above), made it possible to follow the separation of the two substances in the course of their preparation. In the following procedure developed by Thannhauser and his collaborators, 32 the crude ether-insoluble phosphatide mixture is precipitated from glacial acetic acid with a large volume of acetone. After alkaline hydrolysis to destroy the hydrolecithin, sphingomyelin can be prepared in pure form from the precipitate. Lung tissue is used as the starting material since, in addition to containing large amounts of sphingomyelin, it contains only small amounts of cerebrosides. The same procedure may be used for spleen. The large amount of cerebrosides in brain makes the fractionation of the ether-insoluble material from that tissue much more difficult. The modifications necessary for the isolation of brain sphingomyelin have been described by Thannhauser and Boncoddo23 Step 1. Preparation of Crude Sphingomyelin. Fifty pounds of beef lung is minced and washed twice with acetone, filtered, and dried in vacuo at 60 °. The tissue is then ground to a powder and extracted continuously 80 p. A. Levene, J. Biol. Chem. 24, 69 (1916). 81 S. J. Thannhauser and P. Setz, J. Biol. Chem. 116, 527 (1936). 8~S. J. Thannhauser, J. Benotti, and N. F. Boncoddo, J. Biol. Chem. 166, 669, 677 (1946). 88S. J. Thannhauser and N. F. Boncoddo, J. Biol. Chem. 172, 141 (1948).
344
LIHDS AND ST~.ROIDS
[57]
with ether for 3 days. After standing in the refrigerator overnight, the ether extract is filtered over Hyflo filter aid (Johns-Manville). The precipitate is re-extracted with ether in a Soxhlet apparatus for several days to remove any further ether-soluble impurities. The residue is taken up in 1 1. of 9:1 petroleum ether-methanol and filtered. The filtrate is concentrated to a thin sirupy liquid which is precipitated with 1 to 2 1. of acetone. After standing in the refrigerator overnight, the precipitate can be collected by filtration. The precipitate is mainly a mixture of sphingomyelin and hydrolecithin. Yield, 20 to 40 g.; P, 3.8 to 4.0%, of which 40 to 50 % is saponifiable. Step 2. Separation of Sphingomyelin from Bulk of Hydro~cithin. The crude ether-insoluble phosphatide mixture is warmed slightly with 10 vol. of glacial acetic acid. After standing overnight at room temperature, the solution is filtered and the residue is re-extracted with 10 vol. of acetic acid. The pooled filtrates are concentrated to a small volume and then precipitated with 1000 to 1500 ml. of acetone. After standing overnight in the refrigerator the suspension is filtered. The precipitate contains sphingomyelin and hydrolecithin in the proportion 2:1 and is completely free of unsaturated monoaminophosphatides. Step 3. Preparation of Sphingomyelin by Alkaline Hydrolysis. Ten grams of the above material is suspended in a small amount of water and ground to a paste. A total of 200 ml. of 0.25 N N a 0 H is added, and the suspension is shaken at 37 ° for 4 or 5 days. It is then acidified with glacial acetic acid, refrigerated overnight, and filtered over Hyflo filter aid. The precipitate is washed with acetone, followed by ether, and then extracted in a Soxhlet apparatus for 2 to 3 days with ether. The contents of the thimble are dialyzed for 24 hours against running water to remove inorganic contaminants; the dialyzed suspension is filtered over Hyflo filter aid and washed with acetone. The residue is taken up in 9:1 petroleum ether-methanol and filtered. The filtrate is concentrated to a small volume and precipitated with 1000 to 1500 ml. of acetone. Yield of precipitate, 3 to 4 g. Step ~. Removal of Traces of Cerebrosides. The precipitate is taken up in a small amount of 9:1 petroleum ether-methanol and run through an A1203 chromatographic column for the selective absorption of eerebrosides. The sphingomyelin is recovered by precipitation of the effluent fluid with acid and reerystallization from hot ethyl acetate. N, 3.4%; P, 3.58 %; N : P , 2:1; and less than 1% of the total P is saponifiable.
Properties Lung sphingomyelin so obtained is a white crystalline substance which is relatively stable to light and air and is nonhygroscopic. It is soluble in
[57]
PHOSPHATIDES
345
benzene, chloroform, warm alcohol, and hot ethyl acetate but insoluble in ether and acetone. It can be crystallized from hot ethyl acetate and it emulsifies in water easily. Its melting point is 209 °. Sphingomyelin contains two asymmetric carbon atoms and is therefore optically active. Thannhauser et al. 32 report [~]~9 = -~6.25 (4% solution in 1 : 1 chloroformmethanol). This is slightly lower than the values reported by Levene, 3° but the latter's preparations were undoubtedly contaminated with hydrolecithin. Sphingomyelin is capable of forming complexes with metallic compounds such as cadmium chloride and ehloroplatinic acid. The insoluble compound with Reinecke acid has been used for analytical purposes. However, Hack 34 has shown that the sphingomyelin reineckate formed is simply an adsorption complex and not a stoichiometric chemical compound and is therefore unsuitable as an analytical method. The iodine number of the sphingomyelin isolated by Thannhauser is 30 (theoretical = 36), indicating the presence of one double bond. Since sphingosine contains a double bond, only saturated fatty acids can be present. Approximately equal amounts of palmitic and lignoceric acids were found in sphingomyelin prepared from lung or spleen. On the other hand, the component fatty acids of brain sphingomyelin are stearic, nervonic, and lignoceric acids. The acid-base relationships of sphingomyelin are very much like those of lecithin, since they both have a strong acid and a strong basic group. The basic character of sphingosine need not be considered, since its amino group is bound in amide linkage. Sphingomyelin exists as a zwitterion. It shows no buffering power over a wide range of pH's and shows no combination with either chloride or sodium ions. 35 Chain and Kemp 38 determined the isoelectric point of sphingomyelin to be 6.01 This somewhat low value was attributed to the presence of an acidic impurity. P u r i t y . Sphingomyelin preparations should be free of glycerol, free amino nitrogen and saponifiable phosphorus. All the phosphorus of sphingomyelin is resistant to the action of alkali under the conditions described above by Schmidt et al. 7 Nitrogen and phosphorus are present in a 2:1 ratio, in contrast to lecithin, phosphatidyl ethanolamine, and phosphatidyl serine, which have 1:1 ratios. Choline and sphingosine should be the only nitrogen-containing residues present. 34 M. H. Hack, J. Biol. Chem. 166~ 455 (1946). 35 H. N. Christensen and A. B. Hastings, J. Biol. Chem. 136, 387 (1940). 3e E. Chain and I. Kemp, Biochem. J. 28~ 2052 (1934).
LIPIDS AND STEROIDS
[57]
Titration. After cooling to 20 °, pipet a 4-ml. aliquot of the benzene extract into a 5-ml. test tube, and evaporate the benzene to dryness in a tube heater or on a steam bath under a stream of nitrogen. Add exactly 1 ml. of alcoholic thymol blue, taking care to wash down the walls of the tube. By means of a fine capillary, bubble a fine stream of nitrogen or C02-free air through the solution for several minutes in order to remove any carbon dioxide. Then titrate the fatty acids with tetramethyl ammonium hydroxide from a micrometer buret whose tip extends below the surface of the liquid. The end point is reached when the indicator changes from a bright yellow to a persistent yellowish-green tinge. Place a stoppered control tube containing 1 ml. of the acid alcoholic thymol blue in permanence next to the solution being titrated. A reagent blank is run with each set of determinations, the titration value being about 1 #l. Calculations. V = net titration value in microliters; N = normality of N(CHs)4OH; and W = body fluid in milligrams or microliters.
Meq. of fatty acid in aliquot titrated -
V×N
1000
(l)
F a t t y acids in body fluids, usually expressed as milliequivalents per liter, are calculated as follows: V > ( N >(1000 5 Meq./1. = W × ~ (2) Normal plasma values vary from 6 to 20 meq./1. To convert to milligrams of fatty acids per 100 ml., multiply the milliequivalents per liter by 27.72, the average molecular weight of blood fatty acids, according to Stoddard and Drury.**
[57] Preparation and Analysis of Phosphatides 1 By ~/~ARJORIE B. LEES
Assay Methods The quantitative analysis for total and individual phosphatides presents numerous problems associated with the completeness of their extraction, the removal of nonlipid contaminants, hydrolysis procedures, and the specificity of the methods. The complete extraction of the phos1The excellent books by H. Witcoff, "The Phosphatides" (Reinhold Publishing Corp., New York, 1951) and H. J. Deuel, Jr., "The Lipids" Volume I (Interscience Publishers, Inc., New York, 1951) are recommended for further references and discussion of much of the material presented.
[57]
PHOSPHATIDES
329
phatides and the removal of nonlipid contaminants are prerequisites for any method for the determination of total phosphatides (Vol. III [55]). The most commonly used method is the analysis of an adequately extracted, dialyzed sample for phosphorus. This is as accurate as and much simpler than the oxidative method of Blooff or the determination of fatty acids recommended by Artom. 3 Analyses for lipid phosphorus can be carried out by the method of Sperry 4 or by any other suitable procedure. The phosphorus value obtained is converted to phosphatides by means of a factor based on the average phosphorus content of the phosphatides. Although various investigators have used experimentally determined factors, multiplying the lipid P by 25 (based on an average P content of 4%) is adequate for most purposes. The best methods for the analysis of individual phosphatides are those based on a study of their hydrolysis products. Methods dependent on the solubility of the lipids are completely inadequate, since the solubility of the individual lipids is influenced by the presence of other lipids in a mixture. The cephalin fraction, for example, is insoluble in alcohol, whereas the phosphatidyl ethanolamine isolated therefrom is completely alcohol-sohible. After hydrolysis of the phosphatides, a combination of several analytical techniques can be used to determine the amounts of the main components of the mixture. Table I shows the distribution of various chemical components among the principal phosphatides. Phosphatides such as cardiolipin, acetal phosphatides, and phosphatidic acids have been omitted from this discussion, since they either have been isolated only from special tissues or have not been sufficiently characterized to warrant their inclusion here. Brief descriptions of their preparation may be found in Witcoff's book.' The following relationships can be seen from Table I: (1) lecithin and sphingomyelin are the only known choline-containing lipids; (2) the amount of the lecithin plus cephalin fraction is represented by the amount of glycerol present; (3) the total amino nitrogen value gives the sum of the phosphatidyl ethanolamine and phosphatidyl serine fractions. Glycerol is determined by the method of Blix; 5 inositol can be determined microbiologically or chemically;6 methods for the quantitative determination of choline, ethanolamine, serine, and sphingosine are described in Vol. III [59]. In evaluating the analytical results, however, the effect of the minor phosphatides should not be overlooked. 2 W. R. Bloor, J. Biol. Chem. 82, 273 (1929). C. Artom, Bull. soc. chim. biol. 14, 1386 (1932). 4 W. 5/[. Sperry, Ind. Eng. Chem. Anal. Ed. 80, 46 i1938). 5 G. Blix, Mikrochim. Acta 1, 75 (1937). 6 D. W. WooIley, J. Biol. Chem. 140, 453 (1941) ; B. S. Platt and G. E. Glock, Biochem, J. 37, 709 (1943).
330
LIPIDS AND STEROIDS
[57]
Methods based on preferential hydrolysis constitute a slightly different approach to the analysis of individual phosphatides. Schmidt et al. 7 have shown t h a t the P of monoaminophosphatides becomes acid-soluble under conditions where sphingomyelin remains intact. This has been used as the basis for the assay of sphingomyelin described below. Hack s has developed a similar method for blood phosphatides based on the fact t h a t the choline from lecithin can be hydrolyzed whereas t h a t from sphingomyelin remains intact. After acidification and removal of the unreacted TABLE I CHEMICAL COMPOSITION OF THE PRINCIPAL PHOSPHATIDES
P N a-NH~-N ~ NH2-N ~ Choline [ Glycerol Lipid Atom grams Phosphatidyl choline (lecithin) Cephalins Phosphatidyl ethanolamine Phosphatidyl serine Diphosphoinositide Sphingomyelin
Moles
1
1
0
0
1 1 2 1
1 1 0 2
0 1 0 0
1 1 0 0
By the ninhydrin-CO2 method of D. D. Van Slyke, R. T. Dillon, D. A. MacFadyen, and P. Hamilton, J. Biol. Chem. 141, 627 (1941). b By the nitrous acid manometric method of D. D. Van Slyke, J. Biol. Chem. 83, 425 (1929). sphingomyelin, the filtrate is analyzed for P and choline. F r o m these values and the total P, values for lecithin, cephalin, and sphingomyelin can be calculated. I t should be noted t h a t these hydrolysis procedures are not necessarily applicable to all tissues. Brain lipids in particular often behave differently from those of other tissues. For example, Sperry and Brand's hydrolysis procedure for sphingomyelin could not be applied satisfactorily to brain lipids2 Criteria f o r P u r i t y of Phosphatides. The development of adequate criteria for the determination of the degree of purity of phosphatide preparations still awaits future research. Recent studies using countercurrent distribution and chromatographic techniques have made a beginning in t h a t direction. It is known t h a t the individual phosphatides isolated thus far are not single chemical compounds but are mixtures of closely related substances differing at least in their f a t t y acid residues. 7 G. Schmidt, J. Benotti, B. Hershman, and S. J. Thannhauser, J. Biol. Chem. 166, 505 (1946). 8 M. H. Hack, J. Biol. Chem. 169~ 137 (1947). J. Folch and W. M. Sperry, Ann. Rev. Biochem. 17, 147 (1948).
[57]
PHOSPHATIDES
331
It is significant that, as yet, no synthetic phosphatide has been prepared which is identical with the substances isolated from tissues. Since we are not dealing with a homogeneous chemical compound, the determination of physical constants is not sufficiently precise to be of value in a consideration of the purity of a phosphatide sample. Melting point and solubility, for example, are affected by the chain length and degree of unsaturation of the fatty acid residues. At the present state of knowledge the best approach to a consideration of the degree of purity of a phosphatide preparation is by means of chemical analyses for the amounts of impurities present. The following points should be noted: (1) The complete removal of nonlipid contaminants should be ensured by adequate dialysis of the preparation. It is known that lipids can carry impurities with them through lengthy fractionation procedures. (2) The elemental chemical analysis and the amounts of the hydrolysis products obtained should be consistent with the accepted formula for that phosphatide. (3) No hydrolysis products other than those required by the accepted formula should be present; phosphatide samples should be free of carbohydrates and cholesterol. (4) Hydrolysis products should be present in large enough amounts to exclude the possibility of their being contaminants. Proof that a constituent is combined chemically can best be obtained by a study of the kinetics of its liberation by several agents. (5) the N : P ratio, although widely used in the literature, is not a particularly sensitive criterion for purity, since large amounts of contaminants may be present without affecting the ratio significantly. (6) All the nitrogen (or phosphorus) should exist in the form required by the accepted formula for that phosphatide. With the exception of sphingomyelin, which contains both sphingosine and choline, the nitrogen should be present as a single chemical constituent. Some of the above points will be discussed under the individual phosphatides.
Preparation of Lecithin (Phosphatidyl Choline) Most of the methods for the preparation of lecithin depend on the precipitation of its cadmium salt from an alcoholic solution. The method of Pangborn, 1° described below for the preparation of egg lecithin, is a simplification of that of Levene and his co-workers. 11,12 In Pangborn's method the lecithin-CdCl~ complex is broken with a specific combination of organic solvents, after which the cadmium is removed with dilute alcohol (Fig. 1). l0 M. Pangborn, J. Biol. Chem. 188, 471 (1950). ~1 p. A. Levene and H. S. Simms, J. Biol. Chem. 48, 185 (1921). ~2 p. A. Levene and I. P. Rolf, J. Biol. Chem. 72, 587 (1927).
332
LIPIDS A N D
STEROIDS
[57]
Step 1. Extraction and Preparation of Lecithin-CdCl2. T h e yolks of twelve fresh eggs are stirred for a few seconds in a Waring blendor; 200 ml. of acetone is added, and the mixture is stirred for about 30 seconds, transferred to a beaker, stirred thoroughly with 400 ml. more of acetone, and Egg yolks ÷acetone
Soluble
Insoluble ÷95% ethanol
Soluble
Insoluble
50% CdCI:,
Soluble
Insoluble +CHCls:C2H~0H. 1:7, 3 times
Insoluble
Soluble
l
-i-Pelroleura ether:SO% alcohol, 3:10
Lower phase ffi 80% alcohol
Upper phase petroleum ether
I
Re-extract
with 80% alcohol 2 times
7
~oluble
_5
Insoluble /
)~-I-CHCIs:30 % C~HbOH, 1: 1
phase ffi C~H~OH
Lower phase ffi CHCI3
Upper
!
)Dried~-t-et her: acetone, 5:1, 4°
Insoluble
Soluble Lecithin
Fro. I. filtered by suction. T h e filter cake is extracted five times in the blendor with 200-ml. portions of acetone. T h e creamy white yolk powder thus obtained is extracted for 30 minutes in a mechanical shaker with 800 ml. of 95% ethanol and filtered by suction. This alcoholic extract is precipitated with a slight excess of 50 % aqueous CdC12 (approximately 15 ml.). After standing for 1 hour at 4 °, the mixture is filtered by suction and the precipitate is washed twice with acetone while still on the filter.
[~7]
PHOSPHATIDES
333
Step 2. Purification of Lecithin-CdCl~. The precipitate is dissolved in 100 ml. of chloroform. The slightly cloudy, faintly brown solution is poured with constant mixing into 700 ml. of ethanol containing 10 ml. of 50% aqueous CdC12 to prevent dissociation of the lecithin salt. After intermittent shaking for 10 minutes at room temperature, the mixture is filtered by suction. The precipitate is redissolved in chloroform and treated with ethanol and CdC12 as before, except that it is allowed to stand for 30 minutes with frequent shaking. A third chloroform-alcohol precipitation is carried out as above. The cadmium salt is then suspended in 150 ml. of petroleum ether, and to it is added 500 ml. of 80% alcohol previously saturated with petroleum ether and containing 0.1% CdCl~. After vigorous shaking in a separatory funnel, the alcohol layer is drawn off and the petroleum ether layer is re-extracted twice with a total of 100 ml. of the above 80% alcohol mixture per gram of material remaining in the petroleum ether layer (as determined by drying an aliquot). The combined alcohol extracts are concentrated to two-thirds the original volume to remove the petroleum ether. The concentrated lecithin-CdCl~ mixture is left at about - 5 ° overnight, after which it is filtered by suction. Step 3. Removal of Cadmium. The lecithin-CdC12 precipitate is dissolved in 150 ml. of chloroform, an equal volume of 30% ethanol is added, and the mixture is shaken vigorously for 5 minutes. Under these conditions the lecithin double salt dissociates, and the CdCb. is washed out in the dilute alcohol. Four extractions with 30% alcohol should suffice to remove all the cadmium. An aliquot of the aqueous-alcoholic layer may be tested for the presence of cadmium ions with a drop of 5% AgN03. One extra extraction should be carried out to ensure complete removal of the cadmium. Step ~. Final Purification. The chloroform solution is dried by vacuum distillation of the solvent. The lecithin is dissolved in 100 ml. of anhydrous ether, and to it is added 20 ml. of acetone. The mixture is placed at 4 ° overnight, and the finely flocculent precipitate which forms is removed by filtration over a Btichner funnel. The clear filtrate is evaporated to dryness in vacuo, and the purified lecithin is dissolved in absolute alcohol. Yield, 7 grams; P, 4.03%; N, 1.98%; NH2-N, 0.02%; N : P , 1.06:1. Modifications for Tissues Hanahan and Jayko TM have isolated an individual, completely unsaturated lecithin from baker's yeast by a method involving a final purification of the material on an aluminum oxide column. Since their 1~ D. J. Hanahan and M. E. Jayko, J. Am. Chem. Soc. 74, 5070 (1952).
334
LIPIDS AND STEROIDS
[57]
preparation has been well characterized, it should prove particularly useful for enzyme studies. The preparation of lecithin from tissue extracts is more difficult, and each material presents special problems. A summary of Pangborn's modification for the preparation of beef heart lecithin is as follows. Step 1. Removal of Cardiolipin. 13 6.2 kg. of fresh, defatted beef hearts is ground in a meat grinder. The minced tissue is extracted twice at room temperature with 1.2 ml. of acetone per gram of fresh tissue. The acetone is removed by suction; the residue from the second extraction is dried before a fan until the acetone odor is gone. It is ground to a fine powder which is then extracted three times with 2 1. of 95 % methanol for each 300 g. of powder, each extraction being carried out for a week at room temperature with frequent shaking. After the final extraction, the residue is washed with methanol and discarded. As each extract is collected, it is precipitated with 20% aqueous BaC12 and stored at 3 to 6 °. The pooled extracts are centrifuged; the precipitate can be processed for cardiolipin while lecithin is prepared from the supernatant fluid. Step 2. Precipitation of Lecithin-CdCl2 and Removal of Cadmium. The procedure is then essentially as described above. Lecithin is precipitated from its alcoholic solution by the addition of excess 50% aqueous CdC12; the precipitate is taken up in 200 ml. of chloroform and precipitated with 1400 ml. of ethanol a total of three times, after which the cadmium is removed. The cadmium-free chloroform solution is evaporated to dryness and taken up in 180 ml. of anhydrous ether. To the slightly cloudy solution is added 36 ml. of acetone. The precipitate which forms when the mixture is refrigerated overnight is removed by filtration, and the clear filtrate is evaporated to dryness and redissolved in a small amount of alcohol. Yield, 18 g.; NH2-N, 0.25%. Step 3. Further Purification. The alcoholic solution is made alkaline to phenolphthalein with 10 ml. of saturated aqueous Ba(OH)2, the mixture is immediately neutralized with CO2, and 2 ml. of saturated NaCl is added with vigorous shaking to flocculate the precipitate. The precipitate is removed by gravity filtration, and the clear filtrate is reprecipitated with CdC12. The cadmium salt is precipitated twice by pouring its chloroform solution into alcohol and then fractionated once by the petroleum ether-80% alcohol method described for egg lecithin. The cadmium is then removed, and the lecithin is dissolved in ether to which 20 % of its volume of acetone is added. After refrigeration at - 5 ° overnight, the small amount of precipitate is removed by filtration, and the filtrate is dried and dissolved in a small amount of alcohol. Yield, 10.5 g.; P, 4.21%; N, 1.94%; N : P , 1.01; NH2-N, 0.01%; iodine number, 83.7. ~s M. Pangborn, J. Biol. Chem. 161, 71 (1945).
[57]
PHOSPHATIDES
335
Step 4. Purification for Serological Tests. 1~ The above preparation can be separated chromatographically into fractions of different serological activity (in the Wasserman test for syphilis). A glass column (50 X 250 ram.) is packed with Magnesol-Celite. After impregnation of the column with benzene, 500 ml. of lecithin in 15 ml. of benzene is added at the top of the column, which is then developed with 500 ml. of 2% tert-butyl alcohol in benzene. The column is extruded, and a narrow vertical band is streaked with 1% potassium permanganate in 2.5 N NaOH. The zones which thus show are then eluted with absolute ethanol. This procedure can be applied successfully to commercial lecithin preparations or to lecithin-CdCl~ complexes. Residual impurities can also be removed with aluminum oxide columns. 15
Properties Pure lecithin is a whitish, paraffinlike substance which is extremely hygroscopic. When dry it can be ground to a powder, but on taking up water it becomes a waxy, sticky mass. On exposure to light and air the material darkens and develops a disagreeable odor owing to the instability of the fatty acids. No definite melting point can be determined. Lecithin is readily soluble in methanol, ethanol, benzene, ether, petroleum ether, chloroform, carbon tetrachloride, and carbon disulfide. It is also soluble in pyridine, glycerol, and acetic acid. It is, however, insoluble in methyl acetate and acetone. Lecithin may be precipitated from its water emulsion by the addition of acetone, acids, or inorganic salts. Lecithin containing completely saturated fatty acids (hydrolecithin) is insoluble in ether and is found as a contaminant in the sphingomyelin fraction unless special precautions are taken to remove it. Lecithin is easily hydrolyzed by either acid or alkali. Hack, s using normal KOH at 37 ° for 16 hours, found glycerophosphate, choline, and fatty acids among the hydrolysis products. Since migration of the phosphoric acid radical occurs during hydrolysis, the configuration of the naturally occurring substance cannot be determined from the configuration of the isolated glycerophosphoric acid. 16 The best evidence is that most, if not all, of the lecithin occurs naturally in the a configuration. 17 Lecithin probably exists in the form of a zwitterion. Some sort of internal neutralization is to be expected because of the presence of a strongly acidic and a strongly basic group. Lecithin in solution is essentially neutral and has almost no buffering power in the physiological range. The 14F. A. H. Rice and A. G. Osler, J. Biol. Chem. 189, 115 (1951). 15M. Faure and J. Legault-Demare, Bull. soc. chim. biol. 82, 509 (1950). 16j. Folch, J. Biol. Chem. 146, 31 (1942). 17E. Baer and M. Kates, J. Biol. Chem. 17li~79 (1948).
336
LIPIDS AND STEROID)~
[57]
isoelectric point is rather difficult to determine, but the best values place it in the neighborhood of 6.418 (theory = 7.5). 19 P u r i t y . The most sensitive criterion for the purity of a lecithin preparation is the absence of a-amino nitrogen. 2° No base other than choline should be present. Lecithin preparations should have a N: P ratio of 1 : 1; all the phosphorus should be saponifiable under the conditions of the procedure of Schmidt et al.; and theoretical amounts of choline should be obtained after adequate hydrolysis.
Preparation of Cephalin Phosphatides The multiple nature of the cephalin fraction was first recognized by Folch and Schneider. 21 In the method of Folch 22 described below, the crude cephalin mixture from brain is separated into three different fractions on the basis of their differing solubilities in chloroform-ethanol mixtures. Increasing amounts of ethanol are added to a chloroform solution of cephalin, and the precipitates are collected at various concentrations of ethanol. In this manner three fractions can be isolated: (1) diphosphoinositide, which shows relatively low solubility in alcohol; (2) phosphatidyl serine; and (3) phosphatidyl ethanolamine, which is freely soluble in alcohol. The success of the separation of the crude cephalin mixture is dependent on the fact that phosphatidyl ethanolamine is present as the free ampholyte whereas the other phosphatides are in the salt form. In previous procedures the use of HC1 resulted in their conversion to the free acid form which is evidently more difficult to separate. Aside from the preparation described below, probably the only pure phosphatidyl ethanolamine preparation has been that of Rudy and Page. 2a Although analysis shows the presence of phosphatidyl serine in various tissues, it has never been prepared in a pure state from any tissue except brain. Inositol phosphatides have been reported from a variety of sources. Woolley24 isolated an inositol-containing phosphatide from soybeans which he named lipositol. No formula has been proposed for this compound, although galactose, ethanolamine, tartaric acid, saturated and unsaturated fatty acids, phosphoric acid, and inositol have been identified among its hydrolysis products. It is possible that lipositol is a mixture of closely related lipids. ~sH. B. Bull and V. L. Frampton, J. Am. Chem. Soc. 58, 594 (1936). 19H. Fischgoldand E. Chain, Biochem. J. 28, 2044 (1934). 20D. D. Van Slyke, R. T. Dillon, D. A. MacFadyen, and P. Hamilton, J. Biol. Chem. 141, 627 (1941). ~1j. Folch and H. A. Schneider, J. Biol. Chem. 187, 51 (1941). ~ J. Folch, J. Biol. Chem. 146, 35 (1942); 177, 497 (1949). 2s H. Rudy and I. H. Page, Z. physiol. Chem. 198, 251 (1930). ~ D. W. Woolley,J. Biol. Chem. 147, 581 (1943).
[57]
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337
Step 1. Preparation of Crude Brain Cephalin. Fresh ox brains are freed of their membranes, and 100-g. portions of tissue are ground in a Waring blendor with 300 ml. of acetone for 2 minutes. The portions are combined, and acetone is added until there is at least 3.8 ml./g, of tissue. After a few minutes the acetone is removed by suction and discarded. The precipitate is extracted once more with acetone, once with alcohol, and twice with petroleum ether, 4 ml. of each solvent being used per gram of original tissue. The two petroleum ether extracts are combined and dried completely by vacuum distillation. This dried material is then suspended in 200 ml. of ethyl ether for each kilogram of original tissue and allowed to stand in the refrigerator for a day or two. If a clear supernatant does not separate within three days, water is added to the extent of 1% of the volume of ether. If this does not result in the appearance of a clear upper fraction, the ether suspension should be diluted one-fold with ether. Once a supernatant solution amounting to about one-fourth of the total volume is obtained, the suspension is centrifuged and the precipitate is washed twice with cold ether. The combined ether extracts are dried by vacuum distillation at room temperature, and the residue is dissolved in 50 ml. of ether for each kilogram of initial brain tissue. The ethereal solution is placed in the refrigerator overnight, and any precipitate which forms is discarded. The solution is diluted with an equal volume of ether, and 5 vol. of alcohol is added with stirring to precipitate the cephalin. After the mixture stands at room temperature for about 1 hour, a clear supernatant is formed. The precipitate is collected on a Biichner filter and suspended in 100 ml. of acetone per kilogram of original brain tissue. The suspension is shaken for 40 minutes to remove acetone-soluble impurities. After removal of the supernatant fluid by suction, acetone is again added to the precipitate and the procedure is repeated. Yield, 15 g. of a tan cephalin powder per kilogram of initial tissue; C, 55.2 %; P, 4.13 %; N, 1.59 %; NH~-N, 1.51%; a-NH~-N, 0.76%; NH2-N:N, 0.96; P : N , 1.17. Step 2. Fractionation of Brain Cephalin by Chloroform-Alcohol Method. One gram of the crude cephalin preparation described above is dissolved in 8 ml. of chloroform, and to it is added 1.45 times as much alcohol as chloroform by volume. A turbidity develops and, after standing for about 1 hour, the.-~ixture is centrifuged and resolves itself into a viscous underlayer (fractions I and II, inositol phosphatide fraction) and a clear supernatant. The supernatant is decanted, and to it is added 25 ml. of alcohol. The precipitate (fraction III, phosphatidyl serine) which appears is collected on a Bfichner filter and dried. The filtrate is concentrated in vacuo to half its volume and allowed to stand in the refrigerator for 2 or 3 days. The precipitate (fraction IV) is removed by filtration in the
338
LIPIDS AND STEROID
[57]
cold and dried. The filtrate is concentrated to 1 ml., and to it is added 5 ml. of acetone. After standing for 1 d a y in the refrigerator, the acetoneinsoluble precipitate (fraction V, phosphatidyl ethanolamine) is removed by filtration and dried. T a b l e I I gives an analysis of the above fractions. TABLE II ~ ANALYSIS OF FRACTIONS ISOLATED FROM BRAIN CEPHALIN BY CHLOROFORMALCOHOL METHOD
Components C P N Amino N b ~-Amino N ¢ Inositol Iodine No. Ash Yield, g. per 100 g. eephalin
Fraction Fraction Fraction Fraction V I II III (phospha(inositol (inositol (phosphatidyl phospha- phospha- tidyl Fraction ethanoltide), tide), serine), IV, amine), % % % % % 55.0 4.25 1.15 1.15 0.70 6.8 65.0 16.7 22.0
59.0 3.86 1.36 1.36 0.80 3.4 10.0
60.2 3.58 1.62 1.64 1.47 <0.20 39. g 12.8 27.0
63.0 3.60 1.75 1.60 0.60 <0.20 8.0
66.1 3.65 1.78 1.50 < 0.02 <0.20 78.0 2.5 15.0
From J. Folch, J. Biol. Chem. 146, 35 (1942). b By the nitrous acid manometric method of D. D. Van Slyke, J. Biol. Chem. 63, 425 (1929). c By the ninhydrin-CO~ method of D. D. Van Slyke, R. T. Dillon, D. A. MacFadyen, and P. Hamilton, J. Biol. Chem. 141, 627 (1941).
Step 3. Purification of Inositol Phosphatide by Chloroform-Methanol Method. T h e viscous underlayer (fractions I and I I ) is t r e a t e d with ethanol, and the solid precipitate which forms is collected on a Biichner filter and dried. I t is then processed at 4 ° as follows: One g r a m of crude inositol phosphatide is dissolved in 12 ml. of chloroform, and to the clear solution is added 22 ml. of methanol. T h e mixture is shaken for 30 minutes in a shaking machine, after which the precipitate is collected by centrifugation. I t is then redissolved in 12 ml. of chloroform, 22 ml. of m e t h a n o l is added to the solution, and the procedure is repeated as before as long as the s u p e r n a t a n t solution contains material with less t h a n 4.5% P. I f at a n y point the precipitate fails to dissolve completely in chloroform, the insoluble material can be removed by precipitation with 2 col. of ether. I f the material has not been previously dialyzed, twelve
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339
successive precipitations usually suffice, but as m a n y as thirty precipitations m a y be required on a dialyzed preparation. The crude diphosphinositide so obtained contains large amounts of water-soluble contaminants. These m a y be removed by dialysis as described below. Table I I I gives the analysis of the fractions obtained before and after dialysis. TABLE III ANALYSIS OF BRAIN DIPHOSPHOINOSITIDE a
Component
Before dialysis, %
Mter dialysis, %
31.5 0.79 11.08 6.10 0.10
46.0 0.4-0.6 7.0-7.3 <0.05 <0.02 0.1-0.4 0.4-1.8 9.0 2 i. 0 { <0.01 0.64 2.88 0.5-0.6
C N P Inorganic P a-NH2-N NH2-N (after acid hydrolysis) Carbohydrate (as galactose) Glycerol Inositol K Na Ca Mg Yield, g./kg, fresh tissue
1.7 4.9 8.05 3.25 0.30 1.30 1.0
a Data from J. Folch, J. Biol. Chem. 177, 505 (1949). Step 4. Dialysis of Fractions. A 3 % aqueous emulsion of each of the fractions is prepared by shaking 1 g. of material with 30 ml. of water until homogeneous. The emulsion is dialyzed at 4 ° for 4 days against distilled water with several changes of the outside liquid. The undialyzable fraction is then lyophilized, and the fluffy white powder which is obtained is suspended in acetone, collected on a Biichner filter, and dried. Step 5. Purification of Phosphatidyl Serine. 25 The above method results in phosphatidyl serine preparations which are 85 to 90% pure; i.e., between 85 and 90% of the total nitrogen is present as a-amino nitrogen. If lower values are obtained or if slightly greater purity is required, the following procedure is recommended: One gram of the preparation is dissolved in 10 ml. of chloroform, and to it is added 16.5 ml. of absolute ethanol. A turbidity develops which, on standing or by centrifugation, resolves itself into a viscous underlayer and a clear supernatant solution. The supernatant solution is decanted, and to it is added 30 ml. of absolute ~5j. Folch, J. Biol. Chem. 174, 439 (1948).
340
LIPIDS AND STEROIDS
[57]
ethanol. The precipitate which separates is collected and dried. It contains a higher concentration of a-amino nitrogen than does the mother substance. Phosphatidyl serine preparations of greater than 92% purity are not obtained consistently. The main contaminant appears to be phosphatidyl ethanolamine, since all the nitrogen can be accounted for as amino nitrogen. 28 It is essentially free of cerebrosides ( < 0 . 1 % carbohydrate), lecithin and sphingomyelin ( < 0 . 1 % choline), and cholesterol (<0.1%).
Properties of Phosphatidyl Ethanolamine Freshly prepared phosphatidyl ethanolamine is a slightly sticky white powder which becomes dark brown and stickier on standing in the dark in a vacuum desiccator. This change in physical appearance is evidently not accompanied by any observable change in elementary composition. The material contains 1.7% water, which can be removed at 80 ° in vacuo. However, it returns to its original weight on storage in a desiccator over CaC12. As isolated from brain by neutral solvents, it is ash-free. Phosphatidyl ethanolamine, unlike the cephalin mixture from which it is derived, is freely soluble in alcohol. It is also soluble in chloroform, petroleum ether, carbon disulfide, benzene, hot acetic acid, and wet ether. It is insoluble in anhydrous ether and acetone and ~orms an emulsion with water. It is hydrolyzed relatively easily by either acids or alkali. The iodine number of the product isolated by Folch is 78, indicating the presence of two double bonds for each P atom. The few physical measurements available have been carried out on impure preparations and therefore do not warrant discussion. Purity. All the nitrogen of phosphatidyl ethanolamine should be present as amino nitrogen as determined by the nitrous acid procedure of Van Slyke. 26 The presence of serine can be excluded by the absence of a-amino nitrogen. A phosphatidyl ethanolamine preparation should be free of choline and inositol; all the phosphorus should be saponifiable under the conditions of the procedure of Schmidt et a l . f and it should show a N : P ratio of 1:1.
Properties of Phosphatidyl Serine Phosphatidyl serine is a free-flowing white powder with a slight tendency to darken. After long periods of storage in vacuo in the dark at room temperature there is no change in its elementary composition but there is some sort of molecular rearrangement involving a sharp drop of its a-amino nitrogen and primary amino nitrogen content. Storage in chloroform solution in a dry icebox seems to be more satisfactory. Phosphatidyl 26 D. D. Van Slyke, J. Biol. Chem. 83, 425 (1929).
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341
serine, like phosphatidyl ethanolamine, contains a small percentage of water. It is freely soluble in chloroform, ethyl ether, and petroleum ether, but insoluble in ethanol, methanol, and acetone. In the presence of base, phosphatidyl serine forms a stable, concentrated emulsion with water. However, acidification of the emulsion to pH 1.5 results in a complete precipitation of base-free phosphatidyl serine. The process may be reversed by the addition of theoretical amounts of alkali. Phosphatidyl serine is a strongly acidic compound, having one basic group (the - - N H 2 group of serine) and two acidic, groups (the - - C O O H group of serine and one group from phosphoric acid). Therefore, at physiological pH it binds one equivalent of base for each atom of P. This is shown by the fact that phosphatidyl serine isolated from brain by the use of neutral solvents and freed of water-soluble impurities by dialysis contains Na and K. Preparations obtained by the method described above contain much more K than Na, but Ca and Mg are absent. The same preparations also give theoretical values for an equimolar mixture of oleic and stearic acids. Glycerophosphoric acids, L-serine, and fatty acids have been isolated as hydrolysis products of phosphatidyl serine in approximately 1:1:2 proportions. Intact phosphatidyl serine reacts with ninhydrin and with nitrous acid in the same way as does an a-amino acid, showing that both the carboxyl and the amino groups are free. Its inability to react with periodic acid indicates that the hydroxyl group must be combined. Purity. All the nitrogen of a phosphatidyl serine preparation should be present as free a-amino nitrogen; the presence of any other type of nitrogen indicates the existence of impurities. This is the only quantitative method for differentiating phosphatidyl serine .from phosphatidyl ethanolamine, as other criteria listed under phosphatidyl ethanolamine apply to both substances.
Properties of Diphosphoinositide Diphosphoinositide prepared from brain by neutral solvents is an acidic phosphatide which is obtained as a salt of calcium and magnesium. ~7 It is a white, gritty powder which is not emulsified in water and is insoluble in most organic solvents except wet chloroform. It can be converted to the potassium salt which forms a stable emulsion with water but is insoluble in the usual organic solvents. It is possible to obtain a solution of this material in an organic solvent by diluting a 5 % emulsion with methanol or 2:1 chloroform-methanol. The base can be removed by treatment with dilute acid, indicating that the bases are present in a saltlike linkage. The base-free diphosphoinositide is readily soluble in 27j. Folch, J. Biol. Chem. 177, 505 (1949).
342
LIPIDS AND STEROIDS
[57]
water, forming an acid solution which, on titration, uses one equivalent of base per atom of P present. Brain diphosphoinositide is both alkali- and acid-labile. Among the products of hydrolysis, fatty acids, glycerol, and inositol metadiphosphate have been isolated in approximately equimolar proportions. The chemical structure of inositol metadiphosphate has been established by Folch by (1) elementary analysis, (2) isolation of the theoretical amount of inositol, (3) titration to pH 8.7, which shows the presence of two free acid groups for each phosphoryl radical, and (4) a study of the reaztion products with HI04, which shows that the two phosphoryl groups are in the meta position on the inositol. The attachment of the remaining constituents in the molecule is as yet unknown. The nitrogen and carbohydrate are probably present as impurities. The following formula has been postulated on the basis of the experimental evidence, where R and R' stand for unknown radicals:
j0R
HC-0.P0
J\\o
ItCOH
HCOH OR'
HCOH
\J
HC.0.P0
HCOH Purity. Diphosphoinositide preparations should be free of nitrogen and choline; phosphorus and inositol should be present in a 2:1 ratio.
Sphingomyelin (Monoacylsphingosylphosphoryl Choline) Assay Method Principle. The older methods for the determination of sphingomyelin depended either on its solubility, ~8 which gave low results, or on its precipitation as a reineckate, ~9 which gave high results. The analysis for sphingomyelin developed by Schmidt et al. 7 is based on the fact that these compounds are much more resistant to alkali than are the glycerophosphatides. The conditions of the procedure are such that the selective saponification of the glycerophosphatides is brought about. Since sphingomyelin is represented by the difference between the total and the saponi2s E. Kirk, J. Biol. Chem. 123, 623 (1938). 29 B. N. Erickson, I. Avrin, D. M. Teague, and H, H. Williams, J. Biol. Chem. 136, 671 (1940).
[57]
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343
fiable phosphorus, the values can only be considered approximate in cases where the sphingomyelin content of the tissue is small. On the other hand, it is very useful for detecting the presence of small amounts of hydrolecithin which often contaminates sphingomyelin samples. Procedure. Suitable aliquots of the lipid samples to be tested are placed in wide Pyrex test tubes and evaporated to dryness on a water bath. Each sample is emulsified with 5 ml. of N potassium hydroxide at 37 ° for 24 hours. Schmidt recommends continual shaking of the emulsion, but we have not found this to be necessary. The emulsion is precipitated by the addition of 1 ml. of 5 N hydrochloric acid and 5 ml. of 10% trichloroacetic acid. After standing for 1 hour at room temperature (to hydrolyze acetal phosphatides), the samples are centrifuged and filtered. The phosphorus in the original lipid solution and in the clear trichloroacetic acid filtrate is determined. The difference between the two values represents the nonsaponifiable or sphingomyelin phosphorus.
Preparation Sphingomyelin prepared by the usual methods 3°,31 is contaminated with a large proportion of hydrolecithin. The observation that sphingomyelin is resistant to N alkali at 37 °, in contrast to hydrolecithin which is completely hydrolyzed under those conditions (see above), made it possible to follow the separation of the two substances in the course of their preparation. In the following procedure developed by Thannhauser and his collaborators, 32 the crude ether-insoluble phosphatide mixture is precipitated from glacial acetic acid with a large volume of acetone. After alkaline hydrolysis to destroy the hydrolecithin, sphingomyelin can be prepared in pure form from the precipitate. Lung tissue is used as the starting material since, in addition to containing large amounts of sphingomyelin, it contains only small amounts of cerebrosides. The same procedure may be used for spleen. The large amount of cerebrosides in brain makes the fractionation of the ether-insoluble material from that tissue much more difficult. The modifications necessary for the isolation of brain sphingomyelin have been described by Thannhauser and Boncoddo23 Step 1. Preparation of Crude Sphingomyelin. Fifty pounds of beef lung is minced and washed twice with acetone, filtered, and dried in vacuo at 60 °. The tissue is then ground to a powder and extracted continuously 80 p. A. Levene, J. Biol. Chem. 24, 69 (1916). 81 S. J. Thannhauser and P. Setz, J. Biol. Chem. 116, 527 (1936). 8~S. J. Thannhauser, J. Benotti, and N. F. Boncoddo, J. Biol. Chem. 166, 669, 677 (1946). 88S. J. Thannhauser and N. F. Boncoddo, J. Biol. Chem. 172, 141 (1948).
344
LIHDS AND ST~.ROIDS
[57]
with ether for 3 days. After standing in the refrigerator overnight, the ether extract is filtered over Hyflo filter aid (Johns-Manville). The precipitate is re-extracted with ether in a Soxhlet apparatus for several days to remove any further ether-soluble impurities. The residue is taken up in 1 1. of 9:1 petroleum ether-methanol and filtered. The filtrate is concentrated to a thin sirupy liquid which is precipitated with 1 to 2 1. of acetone. After standing in the refrigerator overnight, the precipitate can be collected by filtration. The precipitate is mainly a mixture of sphingomyelin and hydrolecithin. Yield, 20 to 40 g.; P, 3.8 to 4.0%, of which 40 to 50 % is saponifiable. Step 2. Separation of Sphingomyelin from Bulk of Hydro~cithin. The crude ether-insoluble phosphatide mixture is warmed slightly with 10 vol. of glacial acetic acid. After standing overnight at room temperature, the solution is filtered and the residue is re-extracted with 10 vol. of acetic acid. The pooled filtrates are concentrated to a small volume and then precipitated with 1000 to 1500 ml. of acetone. After standing overnight in the refrigerator the suspension is filtered. The precipitate contains sphingomyelin and hydrolecithin in the proportion 2:1 and is completely free of unsaturated monoaminophosphatides. Step 3. Preparation of Sphingomyelin by Alkaline Hydrolysis. Ten grams of the above material is suspended in a small amount of water and ground to a paste. A total of 200 ml. of 0.25 N N a 0 H is added, and the suspension is shaken at 37 ° for 4 or 5 days. It is then acidified with glacial acetic acid, refrigerated overnight, and filtered over Hyflo filter aid. The precipitate is washed with acetone, followed by ether, and then extracted in a Soxhlet apparatus for 2 to 3 days with ether. The contents of the thimble are dialyzed for 24 hours against running water to remove inorganic contaminants; the dialyzed suspension is filtered over Hyflo filter aid and washed with acetone. The residue is taken up in 9:1 petroleum ether-methanol and filtered. The filtrate is concentrated to a small volume and precipitated with 1000 to 1500 ml. of acetone. Yield of precipitate, 3 to 4 g. Step ~. Removal of Traces of Cerebrosides. The precipitate is taken up in a small amount of 9:1 petroleum ether-methanol and run through an A1203 chromatographic column for the selective absorption of eerebrosides. The sphingomyelin is recovered by precipitation of the effluent fluid with acid and reerystallization from hot ethyl acetate. N, 3.4%; P, 3.58 %; N : P , 2:1; and less than 1% of the total P is saponifiable.
Properties Lung sphingomyelin so obtained is a white crystalline substance which is relatively stable to light and air and is nonhygroscopic. It is soluble in
[57]
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345
benzene, chloroform, warm alcohol, and hot ethyl acetate but insoluble in ether and acetone. It can be crystallized from hot ethyl acetate and it emulsifies in water easily. Its melting point is 209 °. Sphingomyelin contains two asymmetric carbon atoms and is therefore optically active. Thannhauser et al. 32 report [~]~9 = -~6.25 (4% solution in 1 : 1 chloroformmethanol). This is slightly lower than the values reported by Levene, 3° but the latter's preparations were undoubtedly contaminated with hydrolecithin. Sphingomyelin is capable of forming complexes with metallic compounds such as cadmium chloride and ehloroplatinic acid. The insoluble compound with Reinecke acid has been used for analytical purposes. However, Hack 34 has shown that the sphingomyelin reineckate formed is simply an adsorption complex and not a stoichiometric chemical compound and is therefore unsuitable as an analytical method. The iodine number of the sphingomyelin isolated by Thannhauser is 30 (theoretical = 36), indicating the presence of one double bond. Since sphingosine contains a double bond, only saturated fatty acids can be present. Approximately equal amounts of palmitic and lignoceric acids were found in sphingomyelin prepared from lung or spleen. On the other hand, the component fatty acids of brain sphingomyelin are stearic, nervonic, and lignoceric acids. The acid-base relationships of sphingomyelin are very much like those of lecithin, since they both have a strong acid and a strong basic group. The basic character of sphingosine need not be considered, since its amino group is bound in amide linkage. Sphingomyelin exists as a zwitterion. It shows no buffering power over a wide range of pH's and shows no combination with either chloride or sodium ions. 35 Chain and Kemp 38 determined the isoelectric point of sphingomyelin to be 6.01 This somewhat low value was attributed to the presence of an acidic impurity. P u r i t y . Sphingomyelin preparations should be free of glycerol, free amino nitrogen and saponifiable phosphorus. All the phosphorus of sphingomyelin is resistant to the action of alkali under the conditions described above by Schmidt et al. 7 Nitrogen and phosphorus are present in a 2:1 ratio, in contrast to lecithin, phosphatidyl ethanolamine, and phosphatidyl serine, which have 1:1 ratios. Choline and sphingosine should be the only nitrogen-containing residues present. 34 M. H. Hack, J. Biol. Chem. 166~ 455 (1946). 35 H. N. Christensen and A. B. Hastings, J. Biol. Chem. 136, 387 (1940). 3e E. Chain and I. Kemp, Biochem. J. 28~ 2052 (1934).