phosphatidylcholine by high performance liquid chromatography: a partial resolution of molecular species

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1 A large-sale purifiation of phosphatidylethanolamine, lysophosphatidylethanolamine, and phosphatidylholine by high performane liquid hromatography: a partial resolution of moleular speies R. S. Fager, S. Shapiro, and B. J. Litman Departments of Physiology and Biohemistry, University of Virginia Medial Shool, Charlottesville. VA 2291 Abstrat Egg yolk phospholipids, on a 1 g sale, were resolved by high-performane liquid hromatography on an 8-m silia olumn with elution by a stepwise hloroformmethanol gradient into homogeneous phosphatidylholine, phosphatidylinositol, phosphatidylethanolamine, and lysophosphatidylethanolamine frations. Within these frations, partial resolution on the basis of fatty ayl side hain omposition was ahieved. Supplementary key words yolk phospholipids preparative hromatography. egg High-performane liquid hromatography of lipids has great potential that has only partly been realized. To date, high-performane liquid hromatography has been used for analytial or small-sale preparative (a fration of a milligram to a few milligrams) separations (1, 2). However, judiious use of this tehnique is potentially of great value in preparative separations of phospholipid lasses on the sale of several grams. It is the objetive of this paper to investigate this tehnique by arrying out familiar separations (3), namely that of egg yolk phospholipids on silia olumn paking, with use of a high-pressure pump and olumns. We have ahieved separation of the egg phospholipid omponents, phosphatidylethanolamine, phosphatidylinositol, lysophosphatidylethanolamine, and phosphatidylholine. Reent researh, direted towards the eluidation of struture - funtion relations in biologial membranes, has demonstrated the important relationship between membrane funtion and membrane fluidity (4). The latter property is related to the hain length and the 74 Journal of Lipid Researh Volume 18, 1977 degree of unsaturation and substitution of the ayl side hains of the omponent phospholipid moleules (5). Information onerning the fluidity harateristis of moleular speies of different lasses of phospholipids has been primarily onfined to syntheti phospholipids with homogeneous ayl side hains. This is primarily due to the diffiulty in synthesizing speifi moleular speies with heterogeneous ayl side hains. An alternative approah is to attempt to frationate natural phospholipids with respet to degree of unsaturation and in large enough quantities to be employed in a range of physial and funtional studies. To this end, we have ahieved a preliminary subfrationation of the moleular speies of the egg yolk phospholipids on the basis of hain length and degree of unsaturation. MATERIALS AND METHODS High-performane liquid hromatography Separation of lipid omponents was ahieved by means of a silii aid paking (Biosil HA minus 325 mesh, Bio-Rad Laboratories, Rihmond, CA) of 2-4 pm partiles in stainless steel olumns under pressure. Columns were onstruted from type 3 16 stainless steel tubing with Lihroma finish ordered to our speifiations from Handy and Harmon Tube Abbreviations: TNBS, 2,4,6-trinitrobenzenesulfoni aid; TLC, thin-layer hromatography; GLC, gas-liquid hromatography; HPLC, high performane liquid hromatography. Downloaded from by guest, on July 17, 218

2 Company (Norristown, PA). A small test olumn was made from.2 in OD X.15 in ID Lihroma finish tubing ut to 1 m with a olumn volume of 12.6 ml; the large olumn was made from.5 in OD x.397 in ID tubing ut to 1 m lengths. Eight suh meter-length preparative olumns were onneted in series. An individual length of preparative olumn had an internal volume of 78.5 ml per segment, 628 ml for the whole system. The fittings and bed support for the test olumn were Swagelok (Crawford Valve and Fitting, Cleveland, OH) 1/4 in to 1/16 in reduing unions (SS-4-6-1) with an inset 2 pm snubber frit. The fittings and bed support for the preparative olumns were Swagelok 1/2 in to 1/16 in reduing unions (SS ) with an inset 5 pm snubber frit. Connetions between the olumns were through standard type 316 stainless steel 1/16 in OD x 1/32 in ID tubing purhased from Tube Sales (Atlanta, GA). The olumns were dry paked by adding small amounts of silia while vibrating and rotating the olumn. Paking of a 1-m olumn was done in about 45 min. After running a large amount of solvent through the olumn at high pressure, the fittings were removed and it was observed that there was negligible settling of the bed; this shows that the simple paking proedure suffies for preparative olumns of partiles in this size range. Solvent delivery was aomplished by means of an alternating piston pump (Waters Assoiates, Milford, MA, Model 6) with flow rate adjustable from.1 mumin to 9.9 mumin and a pressure limit of 6 psi. An air-operated six port loop injetion valve (ACV-6-UHPa-7-C2) from Valo Instruments (Houston, TX) was used to deposit the sample at the head of the olumn. The sample was put into the sample loop using a Gleno Syringe adaptor (Gleno Sientifi, Houston, TX) and the injetion was triggered pneumatially using a Humphrey Tynamite 4-way air valve (62-4E 1, Humphrey Produts, Kalamazoo, MI). Column operation For this investigation a sample loop of 1 ml volume was used. Crude phospholipids prepared from hen egg yolks omprised the sample (6). Preliminary work, done on the small olumn, onsisted of loading approximately 1 mg and 3 mg on separate oasions and varying the solvent onditions. This was done to examine the effets of sample size elution onditions as well as to find reasonable elution polarities for the individual lipid omponents. Initial work was done with glass-distilled solvents (from Burdik and Jakson, Muskegon, MI). However, reagent grade solvents filtered through a.2 pm Millipore filter were shown to be satisfatory and were used in the large-sale separation. Perentage figures for solvent mixtures refer to perent total volume of methanol added to hloroform. Elution was done with a step gradient of hloroform - methanol mixtures. Appearane of partiular omponents was monitored by spotting aliquots of frations on silia TLC (MN Polygram, Silia G without gypsum) plates and staining with ninhydrin or uprous aetate. Frations were spotted and run against standards on silia TLC plates for absolute identifiation (hloroform-methanol-water 65:25:4 (v/v) for phosphatidylholine, lysophosphatidylethanolamine, and phosphatidylethanolamine; and hloroform-methanol-water 9:7:2 (v/v) for phosphatidylinositol) and assessment of purity. The large-sale separation used a sample of 1 g of rude egg yolk phospholipids. The sample loop was filled and the phospholipid deposited on the head of the olumn in hloroform. This proess was repeated until the entire 1 g, estimated on the basis of a phosphate analysis, was deposited. This was the same level of loading that had been employed on the small olumn. Approximately 5-6 ml of hloroform were run through the olumn to elute the arotenoids. Starting polarity was 1% methanol and onditions of polarity were adjusted aording to the appearane or disappearane of partiular omponents in the eluant frations. The flow rate was maintained at a onstant 5 mumin and frations of 15 ml were olleted. Altogether 844 suh frations were olleted, ending at a polarity of 5% methanol. Approximately one additional liter was olleted in five flasks using 75-8% methanol. All frations were overed with nitrogen and stored in the freezer at -2 C. Fration analysis The basi hromatographi profile from the largesale separation is shown in Fig. 1, a graph of total phospholipid onentration vs. fration number, derived from a phosphate analysis (6) on every 5th or 1th fration. Additional information was obtained by determining total amino phospholipid onentration in eah of these frations. Amino group analysis The usefulness of TNBS as a speifi reagent for the olorimetri analysis of amino groups of amino aids and proteins was first desribed by Satake (7). Siakotos (8) later modified the proedure for waterinsoluble ompounds by use of an organi reation medium and demonstrated reativity of phosphatidylethanolamine and phosphatidylserine. The method Downloaded from by guest, on July 17, 218 Fager, Shupiro, and Litman Preparative HPLC of phospholipids 75

3 Elution Solvents as perent Methanol in Chloroform ("/VI Gas-liquid hromatography Frations within the peak of an individual phospholipid lass were seleted for ayl side hain analysis by GLC. Both preparation of the methyl esters of the ayl side hains of the phospholipids in these frations and the hromatography were as previously desribed (9), with the exeption that the olumn was held at an initial temperature of 16 C for 15 min prior to the rise to a final temperature of 21 C at the rate of 6"Imin. Peak areas were used to determine perent omposition of the individual side hain speies in the seleted frations. Fration Number Fig. 1. HPLC profile of the elution of egg phospholipids from a large-sale preparative olumn, as determined by both phosphate and amino group analysis. Crude egg phospholipids, 1 g, were loaded onto a.397 in ID X 315 in olumn formed from eight equivalent-length setions. The olumn was eluted with a hloroform-methanol step gradient in 15-ml frations, at a flow rate of 5 ml/min. The olumn paking onsisted of Biosil HA minus 325 mesh (Bio-Rad Laboratories). desribed here utilizes a detergent, Triton X-1, as a solubilizing medium for phospholipids. Complete reativity of both phosphatidylethanolamine and phosphatidylserine is observed in the detergent labeling system as evidened by TLC of the reation mixture. Amino-ontaining phospholipid (no more than.25 Fmol of amino groups) was dispersed in.8 ml of.4% Triton X-1 in.2 M NaHC3, ph 8.5. A 2-p.1 aliquot of a 1.5% TNBS in aqueous solution was added and the sample was mixed and allowed to inubate in the dark for 3 min at room temperature. After the inubation period,.4 ml of.5% Triton X-1 in 1.5 N HCl was added to the sample, followed by mixing and storage in the dark. Absorbanes should be measured within an hour of aidifiatjon. The absorbane was read at 41 nm, at whih wavelength one obtains 1.13 absorbane units per.25 pmol of amino groups. The absorbane was linear to at least 1.5 absorbane units. The TNBS solution was made up fresh eah week and stored in a brown bottle at 4 C. 76 Journal of Lipid Researh Volume 18, 1977 RESULTS Calulation of theoretial plates (theoretial plates = 16 V2W-2 where V is elution volume and W is peak width) is often used as one riterion for the resolving power of a olumn (1). While this riterion has some drawbaks in evaluating a step gradient elution of the type employed in this study, it still is valuable for omparison with other separation methods. Both the phosphatidylethanolamine and the phosphatidylholine are mixes of heterogeneous diayl speies. Only in the ase of lysophosphatidylethanolamine is the fatty aid analysis a diret identifiation of moleular speies. Therefore, only lysophosphatidylethanolamine serves as an adequate estimate of theoretial plates. The lysophosphatidylethanolamine elution profile orresponded to 6,5 theoretial plates; this is muh better than TLC separations whih are in the range of one hundred to a few hundred theoretial plates. With preisely the same olumn and paking, it is possible to ahieve higher resolving power by lighter loading. In our ase, baseline separation of the phospholipid lasses was obtained at a 1 g loading. Perhaps an even greater loading at the expense of resolving power ould be used without destroying the separation. Using the 1 mg loading of egg phospholipid on the small olumn resulted in late appearane of the phospholipid omponents relative to a simple gravity olumn on silii aid. Phosphatidylethanolamine, the first omponent, did not appear until elution polarity reahed 4% methanol, while phosphatidylholine did not appear until 7 or 8% methanol. The heavier loading of 3 mg aused an earlier elution of the phospholipids, with some loss of resolution. Phosphatidylethanolamine appeared after approximately 3 ml at 2% methanol and phosphatidylholine at 5-6% methanol. The separation was good enough to serve as a basis for saling up to the large preparative olumn. Fig. 1 shows that Downloaded from by guest, on July 17, 218

4 the peak positions in the large-sale preparation agree with the preliminary work. Phosphatidylethanolamine appeared at 25%, lysophosphatidylethanolamine at 4%, and phosphatidylholine at 5% methanol; omplete separation of the phospholipid omponents was ahieved. Phosphatidylinositol eluted as a small peak just before lysophosphatidylethanolamine. An analysis of eah phospholipid lass, with the exeption of phosphatidylinositol, was made by plotting the perent omposition of eah of the individual ayl side hains against the fration number. The early frations of phosphatidylethanolamine were enrihed in polyunsaturated ayl side hains while the later frations were omposed of the more W Fration Number Y Q, ; 2. TI : IO x + II". I I Fration Number Fig. 2. Ayl side hain omposition of frations aross the phosphatidylethanolamine (PE) elution peak. (A) Ayl side hains that inrease in omposition with inreasing fration number. (B) Ayl side hains that derease in omposition with inreasing fration number. t h - E 1 E 7 v.- F Q).- - a ZI , I Fration Number Fig. 3. Ayl side hain omposition aross the lysophosphatidylethanolamine (IysoPE) elution peak. saturated ayl side hains. It is interesting that while the omposition of polyunsaturates was relatively high in frations 225 through 25 and dropped almost to zero in the later frations, the perent total of unsaturated ayl side hains remained approximately onstant aross the phosphatidylethanolamine peak, thus indiating a nearly one to one ratio of saturated to unsaturated ayl side hains per moleule. Although the level of resolution did not allow an absolute identifiation of moleular speies, it is lear that the 18:, 2:4, and 22:6 ayl side hains showed parallel fall off with inreasing fration numbers, while 16:, 18:1, and 18:2 showed a parallel rise with inreasing fration number (Fig. 2). This implies a preferential pairing of the 18:O with the longer polyunsaturated 2:4 and 22:6 while the 16:O pairs preferentially with the 18:l and 18:2. Only 2% of the ayl side hains of lysophosphatidylethanolamine were unsaturated (Fig. 3), whereas 5-55% of the side hains in phosphatidylethanolamine are unsaturated. The presene of the unsaturated fatty aids ould reflet either a mixture of position-1 and position-2 unsaturated isomers of phosphatidylethanolamine or the presene of some perentage of lysophosphatidylethanolamine derived from diunsaturated speies. Phosphatidylholine, although generally less unsaturated than phosphatidylethanolamine, showed the same trend as phosphatidylethanolamine with respet to ontaining a relatively high onentration of polyunsaturated ayl side hains in the early frations, while the later frations ontained more of Downloaded from by guest, on July 17, 218 Fager, Shaptro, and Litman Preparative HPLC of phospholipids 77

5 h z Fig. 4. Ayl side hain omposition of frations aross the phosphatidylholine (PC) elution peak. the mono- and diunsaturated and saturated ayl side hains (Fig. 4). The assoiation of 2:4 and 2:6 with 18: in the early frations and of 16:O with the 18:l and 18:2 in the latter frations is similar to that whih was observed with phosphatidylethanolamine. The perent of unsaturated side hains remained lose to 5% over most of the frations, rising to 55% in the later frations. DISCUSSION The strategy of approah in liquid hromatography varies onsiderably with the type of ompound to be separated and with the purpose of separation. HPLC has been used primarily as an analytial tehnique and, in suh separations, the most important produt is information per unit time, in terms of theoretial plates per unit time at a resolution adequate to separate the omponents of interest. These separations are usually arried out on small partile (5-1 pm) pelliular (silia shell on an inert glass bead) pakings. For preparative separations the onsiderations are somewhat different. The small partile pakings that are best in a small volume analytial olumn are too expensive and diffiult to pak to be pratial in a 5 ml preparative olumn. We have hosen to use a paking at the small partile size range of what has been previously used in low pressure hromatography of phospholipids, and to take advantage of the apabilities of high pressure pumps and olumns by lengthening our system to 26 ft, thereby inreasing the resolution. In doing so we have been able to obtain separations signifiantly better than analytial TLC and at a loading of 1 g. One approah to preparative separation would be to initially employ a lower resolution separation to obtain partially purified omponents, then to further resolve these into pure onstituents at a lower loading. In our separation, we arried out hemial analyses and TLC on every few frations to evaluate the separation. For this reason the off-olumn proedures took muh longer than the olumn separations themselves. Therefore, we have found it most pratial to use baseline separations in a single pass I 1-1, as a riterion for performane may at first seem questionable. However, high loading on the TLC plates, oupled with the hemial speifiity of the sprays employed, allows a good evaluation of hemial purity of eah phospholipid lass. The results obtained here demonstrate the pratiality of employing HPLC as a large-sale method of purifiation of egg phospholipids. A baseline separation was obtained between lysophosphatidyl ethanolamine and phosphatidylholine. No attempt was made to obtain purified sphingomyelin from the rude egg phospholipid mixture. In addition to the good resolution of individual phospholipid lasses, a onsiderable degree of resolution within eah lass was obtained with respet to ayl side hain omposition. As seen in Figs. 2-4, the leading frations of eah phospholipid peak were very high in polyunsaturated ayl side hains, while the later frations were almost devoid of these side hains. Although resolution to the moleular level was not obtained in this study, the parallel trends observed for speifi ayl side hains aross a phospholipid peak were onsistent with the atual pairings observed for moleular speies in egg yolk phosphatidylholine (1 1). HPLC possesses the potential of yielding even higher resolution by running frations from the preparative olumn on a smaller olumn apable of even higher resolution than the preparative olumn, Downloaded from by guest, on July 17, Journal of Lipid Researh Volume 18, 1977

6 with the goal of obtaining resolution at the moleular level within a phospholipid lass. This would be desirable from several points of view. Most importantly it would greatly simplify the analysis of positional distribution of ayl side hains in the phospholipids of various tissues; this proess urrently involves enzymati formation of diglyerides, aetylation of the 3-hydroxyl group, and analysis by GLC and is therefore destrutive of the original phospholipids (12). The virtue of obtaining separation at the moleular level within phospholipid lasses would be the ability to prepare relatively large amounts of the individual moleular speies that ould be used in studies aimed at obtaining information onerning both the physial properties of these speies and their relation to membrane funtion. Several homogeneous, disubstituted phospholipid speies are available ommerially. However, the diffiulty of preparing asymmetrially substituted phospholipid speies has resulted in only a very restrited number of these types of ompounds being ommerially available and then at very high pries. The ability to resolve moleular speies of phospholipids in a preparative proedure would make these pure, natural phospholipid omponents available for general use in membrane researh and greatly extend the useful range of appliation of HPLC.U The authors wish to aknowledge the exellent tehnial assistane of C. MCluney and J. Murphy. This researh was supported by National Institutes of Health Grants EY548 and EY155 and National Siene Foundation Grant GB Manusript reeived 13 Deember 1976 and aepted 5 Ap-ril REFERENCES 1. Aitzetmuller, K The liquid hromatography of lipids: a ritial review. J. Chromatogr. 113: Jungalwala, F. B., R. J. Turel, J. E. Evans, and R. H. MCluer Sensitive analysis of ethanolamine and serine ontaining phosphoglyerides by high performane liquid hromatography. Biohem. J. 145: Spanner, S Separation and analysis of phospholipids. In Form and Funtions of Phospholipids. G. B. Ansell, J. N. Hawthorne, and R. M. C. Dawson, editors. Elsevier Sientifi Publishing Co., New York, N.Y. Chapter Lenaz, G., and G. Curatola Perturbation of membrane fluidity. J. Bioenerg. 7: Chapman, D Phase transitions and fluidity harateristis of lipids and ell membranes. Q. Rev. Biophys. 8: Litman, B. J Lipid model membranes. Charaterization of mixed phospholipid vesiles. Biohemistry. 12: Satake, K., T. Okuyama, M. Ohashi, and T. Shinoda The spetrophotometri determination of amine, amino aid and peptide with 2,4,6-trinitrobenzene 1- sulfoni aid. J. Biohem. 47: Siakatos, A. N Rapid determination of lipids ontaining free amino groups with trinitrobenzene sulfoni aid reagent. Lipids. 2: Litman, B. J Surfae distribution of the fatty aid side hains of phosphatidylethanolamine in mixed phospholipid vesiles. Biohim. Biophys. Ata. 413: Martin, A. J. P., and R. L. M. Synge A new form of hromatogram employing two liquid phases. Biohem. J. 35: Kulsis, A., and L. Maria Determination of the struture of natural leithins. Lipids. 2: Privett,. S., and L. J. Nutter Determination of the struture of leithins via the formation of aetylated 1,2-diglyerides. Lipids. 2: Downloaded from by guest, on July 17, 218 Fager, Shupiro, and Litmun Preparative HPLC of phospholipids 79