heterogeneity of plasma high density lipoproteins€¦ · * all high density lipoprotein...

$: Heterogeneity of Plasma High Density Lipoproteins€¦ · * All high density lipoprotein (HDL)fractions reported abovewereobtained with the number40 rotor. had been removed by repeated$
Journal of Clinical InvestigationVol. 44, No. 3, 1965

Heterogeneity of Plasma High Density Lipoproteins *ROBERTI. LEVY ANDDONALDS. FREDRICKSONt

(From the Section on Molecular Disease, Laboratory of Metabolism, National Heart Institute,Bethesda, W&d.)

The plasma high density lipoproteins, whichare isolated between the densities 1.063 and 1.21in the ultracentrifuge and which correspond toalpha, lipoproteins on electrophoresis, have beenthe subject of far fewer studies than their counter-parts of lower density. In recent years, however,they have served as useful models for examinationof lipoprotein structure and have become of par-ticular interest to this laboratory since the dis-covery of Tangier disease, in which they are con-genitally absent or seriously deficient (1).

When these lipoproteins are distributed along adensity gradient, they seem to form a continuumwithout stepwise compositional changes (2). Sub-fractions isolated over different portions of thebroad density band by which they are definedhave been found to contain similar lipid composi-tion and protein of the same amino acid composi-tion (3). It is also usually accepted that the highdensity lipoproteins (HDL) are antigenicallyhomogeneous (4, 5) and form a single electro-phoretic band.

These data, however, have never been com-pletely reconciled with other evidence that thehigh density lipoproteins may be discontinuouslydistributed over their common density band. Forexample, the separation of HDL into two majorpeaks in the analytical ultracentrifuge, a techniquedeveloped by deLalla and Gofman (6), is a re-producible phenomenon; it has been repeatedlydemonstrated in different clinical situations thatthese fractions may independently vary in concen-tration (7). Preparative ultracentrifuge fractionscorresponding to these two HDL peaks obtainedin the analytical ultracentrifuge have also beenfound to contain protein moieties of quite differentmolecular weights (8). Subsequently, Shore (9)has presented evidence suggesting that HDL sub-

* Submitted for publication September 15, 1964; ac-cepted November 27, 1964.

t Address requests for reprints to: Dr. D. S. Fredrick-son, National Heart Institute, Bethesda, Md. 20014.

fractions may contain different multiples of a basicpeptide, and this has since been partially confirmedby others (10, 11).

Finally, although only one major immunochemi-cal study of high density lipoproteins (12) hasconcluded that they may be antigenically heter-ogeneous, there are other instances in which morethan one form of this lipoprotein would appear tobe present (13-16). Thus, there is both directand indirect evidence of inhomogeneity amonghigh density lipoproteins. The latter is of un-certain character and has required further defini-tion. This is of particular importance in prepara-tion of pure lipoprotein fractions required in cur-rent experimental work.

An intensive study of high density lipoproteins,with particular reference to their immunochemicalidentity, has therefore been undertaken. By usingspecific antisera to HDL, the lipoproteins presentin plasma and ultracentrifugal fractions have beencarefully characterized. It has been demonstratedthat two forms of alpha lipoprotein exist (17).Their nature and relationship to certain trans-formations of the lipoproteins from their nativestate and the manner in which they help to recon-cile discrepancies among older data are subjectsof this report.

Methods

Unless otherwise indicated, blood was collected in0.1% EDTA from normal males and females betweenages 20 to 45 fasted overnight. Either individual plasmasamples or that pooled from ten subjects was used.Analyses and fractionation were begun on the day of col-lection, and the remaining plasma was stored at 40 C.In a few studies the low density lipoproteins (density< 1.063) of fresh plasma were completely precipitatedwith 0.05 ml per ml 0.1 M manganese chloride and 2mg per ml heparin in the cold (18), and the supernatewas used as a source of alpha lipoprotein.

Preparative ultracentrifugation. Lipoprotein fractionswere separated in the preparative ultracentrifuge bythe method of Havel, Eder, and Bragdon (19). Thedensity of plasma of lipoprotein isolates was raised by

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HETEROGENEITYOF PLASMAHIGH DENSITY LIPOPROTEINS

addition of NaCl and KBr (19) and checked by pyk-nometry at 200 C. Samples were centrifuged at 100 Cin the Spinco model L preparative ultracentrifuge at40,000 rpm (ca. 105,000 g). With the no. 40.3 rotor,centrifugation was carried out for 16 hours at density1.063 and 22 hours at densities 1.1 and 1.21. With theno. 40 rotor, the centrifuge times at comparable densitieswere 24 and 48 hours, respectively. As desired, oneor more of four fractions were usually isolated. Theseare subsequently designated as LDL (density < 1.063),HDL (density 1.063 to 1.21), HDL2 (density 1.063 to1.1), HDL" (density 1.1 to 1.21), and the fraction ofdensity > 1.21.

For use as antigen some LDL was prepared as thefraction of density 1.019 to 1.063. The infranate of thismaterial was not further used to prepare HDL.

The lusteroid tubes were sliced through the inter-mediate colorless zone with a standard tube slicer, andthe top and bottom fractions were centrifuged onceagain at each separation density. This was done be-cause of frequent immunochemical evidence of trace con-tamination of HDL by LDL and of HDL fractions byalbumin and other nonlipoprotein proteins. After re-centrifugation none of these contaminants was present inamounts detectable by immunoelectrophoresis or doublediffusion studies except for trace amounts of LDL thatcould not be removed from the HDL fractions by asmany as three recentrifugations. However, precipitationof LDL (18) before centrifugation eliminated all LDLcontamination in subsequent HDL fractionation begin-ning with density 1.1. All fractions were dialyzed at40 C against 40 vol or more of 0.15 M NaCl containing

0.01 M EDTA at pH 6.5 to 7.3. The dialysis fluidwas changed three times during the 24-hour period.

Analytical ultracentrifugation. Lipoprotein fractionsof densities 1.063 to 1.1 and 1.063 to 1.21 were centri-fuged in the Spinco model E analytical ultracentrifugewithin 24 hours of their preparation without dialysis.A double sector cell was employed, one side containingthe 10 to 30 mg per ml of lipoprotein, the other a solu-tion of NaCl-KBr of the same solvent density, provid-ing the reference boundary. The rotor speed was 52,640or 56,100 rpm and the temperature 200 C. The Schlierenpatterns were photographed at intervals of 4 to 16 min-utes for 2 to 3 hours at a bar angle of 65 to 70.

In the analytical ultracentrifuge at density 1.21, HDL(density 1.063 to 1.21) appeared as an asymmetric singlepeak at 16 to 32 minutes. By 48 minutes it had re-solved into at least two components (Figure 1). HDL(density 1.063 to 1.1) centrifuged at either solventdensity of 1.1 or 1.21 appeared as a relatively homogene-ous peak with a small tail of more slowly floating ma-terial apparent with time. 'In the analytical ultracen-trifuge at density 1.1 HDL (density 1.063 to 1.21)separated by 32 minutes into two peaks. One was ofan area equal to that previously determined for theHDL component; the other was a somewhat broaderand sedimenting peak (HDL2). The Schlieren patternswere the same as those reported earlier by deLalla andGofman (6), who introduced the nomenclature HDL2and HDLs to describe these major components of HDLdistinguishable in the analytical ultracentrifuge. Nosmall peak, comparable to their fraction HDL1 (6, 7)was seen, probably because this lower density material

FIG. 1. ANALYTICAL ULTRACENTRIFUGALANALYSIS OF HIGH DENSITY LIPOPROTEIN (HDL)(D 1.063 TO 1.21). Medium density 1.21, 52,650 rpm, concentration 10 mg of lipoprotein proteinper ml, 260 C. An asymmetric single peak can be seen at 30 minutes (left frame); a by-phasic peak is apparent at 60 minutes (right frame).

427

428 ROBERTI. LEVY AND DONALDS. FREDRICKSON

TABLE I

Lipid and alpha lipoprotein composition of plasma samples studied*

Plasma HDL

Cholesterol Phospholipid Cholesterol Phospholipid Protein

mg per 100 ml

Pool A 176 249 48 104 170Subject 1 195 292 63 125 205Subject 2 121 186 36 74 120Subject 3 163 154 50 96 150Subject 4 198 239 47 100 170

* All high density lipoprotein (HDL) fractions reported above were obtained with the number 40 rotor.

had been removed by repeated preparative centrifugation 4° C for 24 hours. The resulting protein precipitate,at density 1.063. containing 10 to 20% of the cholesterol and up to 50%

Lipid analysis. Two-ml samples of plasma lipoproteins of the phospholipid in the starting material, was thenwere extracted in 50 ml of chloroform: methanol (2:1), washed twice with 25 vol of cold redistilled diethyl ether.and the cholesterol (20) and phospholipid (21) contents The washed precipitate readily redissolved in 0.15 Mdetermined. Protein was estimated by the method of NaCl.Lowry, Rosebrough, Farr, and Randall (22). Repre- Immunochemical methods. Preparation of antisera.sentative examples of the total plasma lipid and HDL Antisera to LDL or HDL were prepared in both sheep(density 1.063 to 1.21) lipid concentrations in the samples and rabbits. Lipoprotein fractions used as antigens werestudied are given in Table I. HDL2 contained from mixed in an equal volume of complete Freund's adjuvant25 to 32% of the total HDL cholesterol and phospholipid, and injected intramuscularly. Some fractions were firstand 20 to 25% of the total HDL protein with the concentrated 5- to 25-fold with Carbowax (15). Theresulting average lipid to protein ratio of about 1.1 for final protein concentration of the antigens was 20 toHDL2 and 0.85 for HDLs. The fraction of density 50 mg per ml.> 1.21 contained less than 2 mg per 100 ml cholesterol The total dose, administered in three divided dosesand 10 to 20 mg per 100 ml of phospholipid, or 6 to at intervals of 3 weeks, was approximately 80 mg of10% of the total plasma phospholipid. antigen protein to Cheviot sheep (wt. 40 to 50 kg) and

Partial delipidation. Preparations of HDL and HDLs approximately 30 mg to rabbits (3 to 5 kg).were partially delipidated by injecting them through a Antigen was stored at 40 C and prepared in adjuvantno. 25 needle into 50 vol of pure ethanol: ether (3: 1) at just before injection. Blood was collected 1 week after

TABLE II

Characterization of antisera used for immunochemical study

Reactivity

OtherAnimal Sensitizing antigen LDL HDL Albumin proteins

SheepSI HDL (D 1.063-1.21) + + + -SIA* HDL (D 1.063-1.21) + + - -S1AB* HDL (D 1.063-1.21) - + - -S2 HDL (D 1.063-1.21) + + + +

RabbitsRI HDL (D 1.063-1.21) - + - -R2 HDL (D 1.063-1.21) + + - -R2B* HDL (D 1.063-1.21) - + - -R3 HDL (D 1.063-1.21) + + + -R3A* HDL (D 1.063-1.21) + + - -R4 HDL (D 1.063-1.21) + + + +R5 HDL (D 1.063-1.21) + :E +R6 HDL (D 1.063-1.21) + i +R7 LDL (D 1.019-1.063) + - - -R8 LDL (D 1.019-1.063) + - - -

HorsesHit Whole serum + + + +H2 Whole serum + 4 + +H3 Whole serum + i + +

* Absorbed with excess albumin (A) or LDL (low density lipoproteins) (B).t H1 = Pasteur Institute (223-224) antiwhole serum; H2 = Walter Reed Army Hospital (Leddy) antiwhole serum;

H3 = Hyland Laboratories antiwhole serum.


FIG. 2. IMMUNOELECTROPHORESISOF FRESH PLASMA BEFORE AND AFTER PRECIPITATION OF THE LOW DENSITYLIPOPROTEINS (LDL). In the left hand slide the trough contains antiserum SA (Table II); on each of themiddle and right hand slides, one trough was filled with antiserum R2 and the other with antiserum R7 (anti-LDL). All precipitation lines on this and succeeding figures are unstained. All the lines shown here were sub-sequently shown to stain for lipids.

the final dose and the serum frozen after the additionof 0.01%o merthiolate. Each antigen was carefully char-acterized by immunoelectrophoresis before use. Thelipoprotein fractions used as antigen for Si, S2, and R1through R. all were derived from pool A (Table I).With each antiserum the precipitating antibodies wereshown by immunoelectrophoresis to be almost entirelyG immunoglobulins (7 S globulin) (23). Certain ofthe antisera were partially purified by sodium sulfateprecipitation by the method of Kekwick (24).

Horse antiwhole human serum' and commercial horseantiwhole serum, rabbit anti-beta lipoprotein, and anti-albumin2 were used.

Characterization of antisera. HDL (density 1.063 to1.21) used as antigen produced adequate anti-HDL titersin both of two sheep and four of six rabbits. All ofthe eight antisera are characterized in Table II. Sevencross-reacted with LDL. Several also formed precipi-tation lines with albumin, and two reacted with otherunidentified proteins. Four rabbit and one sheep antiseracould be absorbed with albumin or LDL leaving suf-

1 Obtained from Walter Reed Army Hospital (Leddy)and the Pasteur Institute (223-224).

2 Purchased from Hyland Laboratories, Los Angeles,Calif.

ficient anti-HDL titer for use in the experiments to bedescribed. LDL produced antibodies of relatively highertiter that did not appear to cross-react with HDL orother proteins. Pure HDL antisera were thus verydifficult to produce, presumably because the antigencould not be freed of undetected LDL, which is so muchstronger in provoking antibody response. Antisera,such as R2 (Table II), however, could be freed of allanti-LDL activity by absorption without appreciabledecrease in anti-HDL titer.

Hyland antialbumin and anti-LDL were relativelypure. They did not react with HDL fractions andgave sharp precipitation bands with albumin and LDL,respectively. All the antihuman sera contained anti-bodies to both HDL and LDL. The Pasteur antiserum(H1, Table II) gave the sharpest precipitation line withalpha lipoprotein at serum concentration and was usedfor most experiments with antihuman serum. The othersera were used only to check ultracentrifugal fractionsfor nonlipoprotein impurities.

Absorption of antibodies was carried out at equivalenceas determined by serial dilution using the double dif-fusion tube method of Preer (25). The precipitateswere allowed to stand for 5 to 10 days before absorbedantisera were harvested.

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ROBERTI. LEVY AND DONALDS. FREDRICKSON

Agar diffusion. Ouchterlony plates or Preer tubes of1 mmi.d. were made with 1% Difco special noble agar insaline. Plates with wells requiring 5 to 10 /Al or 40to 50 pAl of antigen or antibody were used. They wereallowed to develop at 40 C and observed for at least2 weeks. The Preer tubes were sealed after preparationwith molten paraffin and allowed to develop at roomtemperature. The appearance of precipitin bands andtheir location in relationship to the antigen-agar boun-dary were plotted daily for 10 days.

Immunaoelectrophoresis. The technique of Grabar andWilliams (26) as modified for microscope slides byScheidegger (27) was used for immunoelectrophoresis.Standard microscope slides were coated with a thinfilm of 2%o Difco special noble agar in 0.05 Veronalbuffer, pH 8.2, and allowed to dry. Two ml of theagar in buffer was then applied in a coat of uniformthickness. To assure uniform water content, plateswere always used within 2 hours of preparation. Thewells were filled with 2 to 3 ,sl of antigen, and elec-trophoresis was carried out in 0.05 M Veronal bufferat pH 8.2 using constant current (58 ma) for exactly40 minutes. Proteins were either precipitated immedi-ately after electrophoresis with 2%o acetic acid or allowedto diffuse against 50 to 60 /Al of antiserum for 24 to72 hours at room temperature in a high humidity cham-ber. Slides were then washed overnight with saline,followed by distilled water, dried, and exposed to afreshly prepared saturated solution of oil red 0 in 60%oethanol at 400 C for at least 12 hours and then counter-

stained for protein with SF light green for 2 hours.Photographs of the immunoprecipitation lines weremade before washing, after oil red 0 staining, andagain after counterstaining.

Protein analyses. Ultracentrifugal fractions of LDL,HDL, HDL2, and HDL3 were extracted with ethanol:ether (3:1) for 18 to 24 hours at 24° C (3). The pre-cipitate was washed twice with ethanol and once withether. Portions of the precipitate were sealed in an oxy-gen-free atmosphere and hydrolyzed at 1100 C for 24hours in 6 N HCl. Some of the delipidated proteinswere oxidized with performic acid for 1 hour and werethen lyophilized before acid hydrolysis (28). Hydroly-sates representing 0.5 to 5 mg of protein were analyzedfor amino acid content with the Spinco Moore-Steinanalyzer. Quantification was standardized using knownreferences (29). Two of the plasma fractions wereanalyzed four times each, and the amounts of individualamino acids obtained on each run were in agreementwithin 3%o. Amino terminal acids were determined bythe dinitrophenol method of Levy (30).

Results

Heterogeneity of alpha lipoprotein. Immuno-electrophoretic studies. Fresh, whole plasma orplasma from which all low density lipoproteinshad been removed by precipitation with polyanionsusually yielded on immunoelectrophoresis a single

FIG. 3. IMMUNOELECTROPHORETICPATTERN OF PLASMAAND THE ULTRACENTRIFUGAL FRACTIONS REACTED WITH

ANTISERA SlA. All fractions except LDL and the 1.1 to 1.21 (HDLs) fraction are at plasma concentration. LDLand the 1.1 to 1.21 fraction represent threefold increases from plasma concentration. All the precipitation linesseen here were subsequently shown to stain for lipid.

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FIG. 4. IMMUNOELECTROPHORETICPATTERN OF PLASMA AND THE TWO HDL SUBFRACTIONSAFTER PRECIPITATION WITH ANTISERUM RSA. Concentrations of HDL2 and HDL are identicalto those in the previous figure. All visible precipitation lines were shown to stain for lipid.

precipitation arc attributable to alpha lipoproteinwith any of the eight anti-HDL antisera describedin Table II (Figure 2). However, under ap-propriate conditions and especially in certain ultra-centrifugal fractions, heterogeneity of the alphalipoproteins became apparent with all of the anti-sera. As shown in Figures 3 and 4 a single broadarc with anti-HDL serum was present in freshwhole plasma and in the ultracentrifugal fractioncontaining HDL2 (density 1.063 to 1.1). There

was in addition a second band of slower migrationin ultracentrifugal fractions of HDL (density1.063 to 1.21) and HDL3 (density 1.1 to 1.21).Only the second band was present in the fractiondensity > 1.21.

Both of these precipitation bands were lipidstaining, although the second band was occasion-ally only faintly so. The positions of these bandswere relatively constant, the first migrating withand just behind the albumin, the second in the

431


alpha,-alpha2 region. With the antisera, onlythe first band could be detected in HDL2, but bothbands were always detected in HDL3. Only thesecond band was ever detected in the fractiondensity > 1.21. As shown in Figures 3 and 4and subsequently in Figures 5 and 6, these arcsblended completely, crossed, or appeared as sev-eral interrupted lines depending upon the anti-serum used. The precipitation lines obtainedwith HDL (density 1.1 to 1.21) in Figures 3 and4 represented the extreme in complexity obtained,and these were observed only with two antiseraand with concentrations of HDL3 considerably inexcess of that in plasma. The small precipitationlines close to the junction of the major bands wereinterpreted as also representing reactions betweenantisera and the HDL antigens and not unrelatedcontaminants. They were not seen when only oneor the other major band was present alone as in

concentrated preparations of HDL, or the frac-tion of density > 1.21. Furthermore, all the linesstained for lipid, and they were removed by ab-sorption along with the major bands (see below).The multiplicity of lines is considered to reflectthe fact that the HDLwas composed of very simi-lar but not identical antigens. All of the anti-genic preparations used for antibody productioncontained HDL over the density spectrum from1.063 to 1.21. It appears that a family of anti-bodies was produced, its members differing inreactivity to certain antigenic sites, only some ofwhich were common to HDL throughout its den-sity spectrum. It was concluded that there wereonly two major antigens in HDL, these beingdetected with all the antisera.

Recombination and absorption studies. Recom-bination of the lipoprotein fractions with eachother or with plasma (Figure 5) confirmed that

FIG. 5. IMMUNOELECTROPHORESISOF PLASMA BEFORE AND AFTER ADDITION OF AN EQUAL VOLUMEOF A SOLUTIONCONTAINING EITHER HDL2, HDL8, OR THE FRACTION OF D > 1.21 AT PLASMA CONCENTRATION. Antiserum SlAwas used. All precipitation lines stained subsequently for lipid.

432


FIG. 6. IMMUNOELECTROPHORETICPATTERN OF ALBUMIN, LDL, AND HDL WrTH ANTISERA H1.

the alpha lipoprotein in whole plasma and that inHDL2 was identical, and different from the sec-ond more slowly migrating band. By similarcombination experiments (Figure 6), it couldeasily be demonstrated that neither of these bandswas related to either albumin or lipoprotein ofdensity 1.019 to 1.063 (LDL).

With Preer tubes (25), the titers of antiserumSlAB to each of the alpha lipoprotein bands weredetermined separately with a sample of HDL2,containing only the faster migrating band, and asample of fraction > 1.21 that contained only theslower migrating band. This antiserum easilydiscriminated both bands in HDL3. An amountof HDL2 slightly in excess of equivalence wasthen added to SlA. This absorbed antibody wasthen tested against whole plasma and several

HDL fractions by immunoprecipitation, double-diffusion, and immunoelectrophoresis. No reac-tions were now obtained with either plasma orHDL2. Precipitation was obtained with HDL3and the density > 1.21 fraction, which provedonly to represent the slower migrating band onimmunoelectrophoresis.

Similar absorption of antiserum SlAB with thedensity > 1.21 fraction in amounts slightly inexcess of equivalence left reactivity to plasma,HDL2, and HDL3. This was shown to repre-sent only the fast migrating band on immuno-electrophoresis. No reaction was obtained withthe density > 1.21 fraction. Quantitativelyabout two-thirds of the reactivity of antiserumSlAB could be removed with either HDL2 or the> 1.21 fraction. Results of similar absorption

433


experiments using other antisera varied depend-ing on their ability to discriminate between thetwo forms of alpha lipoprotein.

Nomtenclature. On the basis of the above ex-periments, the two forms of alpha lipoproteindistinguishable by immunoelectrophoresis havebeen designated as alpha LPA, the band of greatermigration seen in plasma, HDL, HDL2, or HDL3;and alpha LPB, the second band present only inHDL3 and the density > 1.21 fraction (Figure7). This nomenclature incorporates the designa-

tion "LP" to differentiate the lipoprotein fromtthe delipidated protein, alpha P.3

Diffusion and precipitation studies. Smallamounts of alpha LPB could be detected in freshplasma by immunoelectrophoresis if the plasmawas concentrated 5- to 10-fold by polyethyleneglycol (15) or triple-loading of the antigen wells.

3 In a preliminary communication (17), alpha LPB wasreferred to as alphaD, a simpler term, but offering lessflexibility for possible new forms of alpha lipoproteinthat conceivably may be demonstrated in the future.

FIG. 7. IMMUNOELECTROPHORETICPATTERNSOBTAINED WITH ANTISERUM R1 AND LIPOPROTEINFRACTIONS HDL2 (D 1.063 TO 1.1) AND DENSITY GREATERTHAN 1.21 UNDERGOINGELECTROPHORE-SIS SEPARATELY (LEFT HAND SLIDE) AND IN COMBINATION (RIGHT HAND SLIDE). HDL2 con-tains only the form designated alpha LPA, and the density greater than 1.21 fraction containsonly the form designated alpha LPB. All the lines shown here stain for lipid.

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FIG. 8. IMMUNOELECTROPHORETICPATTERN OF HDL2 (ALPHA LPA) AND ITS PARTIALLYDELIPIPATED PRODUCT. Antiserum SlA was used. Both lines shown here stain for lipid.

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Usually very small amounts of this second alphaband could be detected in unconcentrated plasmaby Ouchterlony plates or especially by the methodof Preer. Even with these more sensitive tech-niques, only one line was seen with HDL2 usingany of the antisera, emphasizing the immunologichomogeneity of this lipoprotein fraction.

With either the Preer or Ouchterlony tech-niques, repetition of the absorption studies withantiserum SlA, described above, using immuno-electrophoresis, again demonstrated that alphaLPA and alpha LPB differed in some antigenicsites for this antiserum.

As could be predicted from immunoelectropho-resis, the two forms of alpha lipoprotein couldalso easily be distinguished by differing mobilityon simple electrophoresis on agar.

Conversion of alpha LPA to alpha LPB. Theabsence of significant amounts of alpha LPB fromfresh serum and its obvious appearance in certainof the ultracentrifugal fractions implied that thelatter process was capable of producing alpha LPB,quite probably through the conversion of some ofalpha LPA.

The appearance of alpha LPB during ultracen-trifugation proved to be dependent upon the ex-posure of lipoprotein to a centrifugal field andnot to the other manipulations attending the rou-tine preparative procedure. Serum brought to adensity of 1.063 by addition of NaCl-KBr, al-lowed to stand at 150 C for 1 week, and then

TABLE III

Amino acid compositions of the proteins of serumLDL, HDL, and HDL subfractions*

LDL HDL HDL2 HDL8

Aspartic 100 100 100 100Threonine 56 58 58 60Serine 74 84 84 81Glutamic 118 244 244 239Proline 40 56 59 59Glycine 45 54 51 52Alanine 59 93 93 93Cystinet 7 7 6Valine 52 68 66 65Methionine 13 10 12 10Isoleucine 53 7 6 6Leucine 108 161 159 160Tyrosine 33 39 39 40Phenylalanine 49 41 41 40

* All values are expressed as moles relative to asparticacid taken as 100. LDL = D 1.019 to 1.063.

t Cystine determined as cysteic acid after pretreatmentof hydrolysate with performic acid.

dialyzed against saline containing 0.01 M EDTAcontained no alpha LPB detectable by immuno-electrophoresis. In contrast, alpha LPB becamereadily apparent at serum concentration in theinfranate after a single 16- to 24-hour centrifuga-tion at density 1.063. Furthermore, repeatedcentrifugation at density 1.21 of either HDL orHDL2 yielded additional alpha LPB in the in-franate of each successive run indicating continu-ous production of this form of the lipoprotein dur-ing ultracentrifugation.

Storage, as well as ultracentrifugation, wasfound to lead to the production of alpha LPB.Although fresh serum or plasma obtained withEDTA, sodium oxalate, or heparin as anticoagu-lant contained little or no detectable alpha LPB,it became increasingly demonstrable during stor-age of the unconcentrated samples over a periodof 2 to 10 days. Storage of plasma in EDTAminimized the generation of alpha LPB.

Alpha LPB was also produced by repeatedfreezing and thawing between 40 and - 20° C orby addition of urea to plasma in a final concentra-tion of 8 M. As alpha LPB became apparent, thetiter of alpha LPA decreased. As noted earlier,the supernate remaining after removal of LDL byprecipitation (18) contained only alpha LPA(Figure 2), but the B form was then easily pro-duced by either simple storage of the supernatewithout EDTA, or by freezing and thawing. Thisexperiment provided further proof that alphaLPB was derived from alpha LPA and not fromother lipoprotein precursors.

Final proof of the conversion of the A formto the B was provided in the following experi-ment, the results of which are shown in Figure 8.An HDL2 fraction, containing no alpha LPB, waspartially delipidated with ethanol: ether (3: 1)and washed with ether. The resulting materialwas completely soluble in 0.15 M NaCl. It wassubjected again to immunoelectrophoresis andconsisted almost exclusively of alpha LPB. Par-tial delipidation of the fraction 1.1 to 1.21, in thesame way, again eliminated most of the alphaLPA with a concomitant gross increase in theamount of alpha LPB. The slower migratingmaterial produced by delipidation of alpha LPAwas recombined with plasma, HDL,, HDL3, andthe fraction density > 1.21. Both immunoelec-trophoresis and double diffusion of these mixtures

436


on Ouchterlony plates indicated that the delipi-dated product was immunochemically identicalto alpha LPB.

Protein analyses. In Table III are presentedthe relative amino acid compositions of the lipo-protein fractions LDL, HDL, HDL2, and HDL3.The values given for each fraction represent themeans obtained on 3 to 5 separate preparationsof each fraction. These, as well as separate de-terminations on the same fractions, all agreedwithin 3%c. The amino acid compositions ofthese HDL fractions were indistinguishable anddifferent from those previously published forLDL and albumin (3, 31). The basic aminoacids, arginine, histidine, and lysine are not

shown in Table III. They were determined once

on a single sample of each fraction. The con-

tents of these amino acids were indistinguishablein HDL and its fractions. No attempt was madeto correct for possible losses of amino acids dur-ing hydrolysis, nor was there an attempt to

quantify tryptophan or amide ammonia. As previ-ously noted by others (3, 9, 11, 32), the HDLfractions all contained amino terminal asparticacid with trace amounts of serine and threonine.In view of the similarity of the amino acid analysesof the HDL fractions, the peptides contained inalpha LPA and LPB are very probably identical.The analyses of HDL amino acids (Table III)are comparable to those previously published ex-

cept for discrepancies in cystine content (3, 9-11,33). For the latter our results are in agreementwith the data of Shore and Shore (33), who useda titration method for determining sulfhydrylgroups, but in disagreement with the data ofScanu and Hughes (3) and Sanbar and Alau-povic (10), which showed little or no cystine.This is probably explained by our pretreatment

of protein hydrolysates with performic acid con-

verting the cystine and cysteine present to themore stable cysteic acid before acid hydrolysis(28).

Discussion

Two forms of alpha lipoprotein, designated Aand B, have been demonstrated. The native stateof this lipoprotein in plasma appears to be almostentirely alpha LPA. As plasma ages, undergoesultracentrifugation, or is otherwise manipulated,alpha LPB appears. It is distinguished from

alpha LPA by differences in electrophoretic mi-gration, immunochemical behavior, and flotationin the ultracentrifuge. Proof has been presentedthat alpha LPB can be derived from alpha LPAby partial delipidation, but there is other evidencethat this transformation may involve more thansimple loss of lipid.

The importance of these findings lies particu-larly in three areas: First is the practical prob-lem of having to deal with several forms of alphalipoprotein in any immunochemical studies ofplasma lipoproteins, particularly those aimed atdefining the purity of lipoprotein isolates. Thesecond is the implication that use of the ultra-centrifuge to isolate and quantify high densitylipoproteins is associated with molecular rear-rangements that give rise to heterogeneity thatprobably does not exist in normal plasma. Fi-nally, the observed transformation of alpha lipo-protein correlates with other data relevant to thepeptide portions of high density lipoprotein insuch a way as to offer further insight into thestructure of lipoproteins.

The eight different antisera employed in thisstudy varied in their specificity for the A and Bforms of alpha lipoprotein. All, however, whenused with immunoelectrophoresis, distinguishedthese two forms. Most of the antisera showedonly partial cross-reactivity with each by eitherimmunoelectrophoresis, double diffusion, or dif-ferential absorption. In assessing the validity ofthese findings it has been necessary to reconsiderwhy, if the differences between alpha LPA andalpha LPB now seem apparent, has heterogeneityof alpha or high density lipoproteins not beenmore widely recognized in many previous im-munochemical studies of these lipoproteins. Inreviewing these in light of the present experi-ments, it is impressive how much evidence ofheterogeneity has actually been present, and howclosely it seems to corroborate the present demon-stration of more than one form of alpha lipo-protein.

Aladjem, Lieberman, and Gofman (12) madethe first immunochemical studies of HDL andconcluded that these lipoproteins were not (anti-genically) homogeneous. Interestingly, theyfound evidence of antigenic differences betweenthe centrifugal subfractions HDL2 and HDL3.The only other study employing a specific anti-

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HDL serum was that of DeLalla, Levine, andBrown (5). Although their experiments are of-ten cited as proof of the homogeneity of HDL,their data are actually more consistent with theirsingle antiserum having been primarily reactivewith what is now called alpha LPB. Its titer ofreactivity for alpha lipoprotein was much higherwith aged plasma, HDLfractions, and the fractionof density > 1.21 than with comparable amountsof fresh plasma. They also obtained precipitationlines showing only partial identity between plasmaand ultracentrifugally isolated HDL fractions inreacting with their antiserum.

Heterogeneity of alpha lipoprotein can also beseen in most published experiments using im-munoelectrophoresis with antiwhole human sera.This was recognized by Ayrault-Jarrier, Levy,and Polonovski in their work (15), but it alsoappears to be present in the patterns publishedby Uriel and Grabar (13) and Grabar and Bur-tin (14). If one assumes that the absolute mi-gration rates of the three lipid-staining bands ob-tained by Uriel and Grabar (13) differed fromours due to differences in water content of theagar (34), their "rho-lipoprotein" and "lipal-bumin" lines represent alpha LPA and LPB,respectively. Neither line was present in theirprecipitate of LDL obtained by polyanions, thelatter yielding only a single lipoprotein band,which they called "alpha." Burstein and Fine,using a technique that precipitates some of thealpha lipoprotein from material first freed ofLDL, have also recently found two precipitation1)ands (16).

Scanu, Lewis, and Page also obtained two im-munoelectrophoretic bands with HDL (4), butattributed one of these to albumin contamination.It is interesting how closely their published pat-terns resemble those we have obtained with thetwo forms of alpha lipoprotein.

Today the most widely used techniques forthe preparation and quantification of lipoproteinsdepend upon the ultracentrifuge. Column chro-matography has been attempted without demon-strated success (35). Precipitation methods withhigh molecular weight substances, which quitesuccessfully permit separation of pure low densitylipoproteins (18, 36), have so far proved capableof isolating only a small fraction of the total alphalipoprotein in plasma (16). There still is, there-

fore, no method for isolating large amounts ofhigh density lipoproteins that does not involvehigh speed centrifugation at least as a purificationstep.

The present studies are disturbing in theirdemonstration that ultracentrifugation is associ-ated with alterations in high density lipoproteinsthat affect both their structure and quantitativerecovery. The native form of HDL, alpha LPA,is progressively converted to a lipoprotein form ofhigher density (alpha LP11) with the loss of someto the infranate of the accepted density limit of1.21. Such losses probably have been responsiblein part for the tendency of concentrations of highdensity lipoproteins to be lower than that of theircounterparts isolated by Cohn fractionation andelectrophoresis (37). The conversion of alphaLPA to alpha LPB also probably accounts formuch of the great variation in relative concentra-tions of HDL subfractions reported in the litera-ture (2, 3, 7, 19, 38).

The two forms of alpha lipoprotein have beenshown to bear a constant relationship to the twopeaks into which HDL separates in the analyticalultracentrifuge by the technique of deLalla andassociates. HDL., (density 1.063 to 1.1) repre-sents only the A form, whereas HDL3, the frac-tion of greater density (1.1 to 1.21), containsboth A and B. Theoretically the relative amountsof HDL2 and HDL3 can be expected to vary withthe degree of conversion of alpha A to B. It isinteresting in this regard to compare analyses ofHDL2 and HDL3 obtained in the analytical ultra-centrifuge. Two such studies, in two similar pop-ulations with similar total HDL concentrations,have been reported in the past few years from theDonner Laboratory (7, 38). The ratios of HDL2to HDL3 in the two sets of data are very different.There may be other possible explanations forthese differences, but the fact remains that con-version of alpha lipoprotein during ultracentri-fugation must be taken into account in assessingthe significance of HDL subfractions. It is pos-sible that the conventional subfractions, HDL2 andHDL3 (5), may be "artifactual" in the sense oftheir being more related to the transformability ofalpha LPA than to any specific metabolic influ-ences.

The nature of the transformation of alpha LPAto alpha LPB can only be inferred from the pres-

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ent data. B certainly contains relatively lesslipid than A, as judged from their comparativeflotation characteristics and lipid staining, and asconfirmed by lipid analyses before and after con-version of A to B by treatment with ethanol:ether. The density range in which A and B arefound is broad and overlapping, suggesting thatthe lipid contents of the A and B molecules areboth variable and frequently similar. This sug-gests that other differences also probably existbetween these two forms.

A possible difference in the protein componentof the two forms of alpha must be considered.As indicated, the amino acid compositions of thepolypeptide forms A and B are indistinguishable.It may be presumed that differences in their elec-trophoretic mobility and antigenicity therefore canmost likely be due to some change in the structureof the protein of the lipoprotein. From previouswork it is possible that such a structural changecould be the presence of different multiples of anidentical peptide subunit.

Shore (9) has shown that the protein containedin HDL subfractions of density 1.093 and 1.149had the same amino acid analyses and end groups.The protein in density 1.093 molecules had twicethe molecular weight of that in the 1.149 frac-tion and twice the number of amino terminalgroups. He postulated that the protein in thelighter fraction was a dimer of that in the heavierfraction. Scanu, Lewis, and Bumpus (11) andSanbar and Alaupovic (10) have found a singleprotein peak after delipidation of HDL over theentire 1.063 to 1.21 density range. The molecularweight of this protein was calculated to be about75,000 or comparable to that of the monomer ofShore (9). Sanbar and Alaupovic found thatthis delipidated protein separated into two peakson standing. The second peak appeared to be anaggregate of the first and could readily be con-verted to the first peak in the presence of 1 to 4 MIurea or increased pH (10). More recently Shoreand Shore (33) have presented evidence that thedelipidated density 1.125 to 1.20 lipoproteins canbe chemically divided into identical repeatingprotein subunits of about 36,000 mol wt.

The report of Scanu and Hughes (3) thattheir delipidated protein of 75,000 mol wt mi-grated upon electrophoresis more slowly on agarand more rapidly on starch gel than did alpha

lipoprotein is consistent with the relative mobilitiesof alpha LPB and LPA in these same media.

It is considered that alpha LPB could representa "monomer" form of alpha lipoprotein and alphaLPA a "dimer" or larger aggregate than alphaLPB- It will be necessary to obtain the molecularweight of the protein in each form before thishypothesis can be proved. How aging and ex-posure of plasma to a high centrifugal field re-move lipid from the lipoprotein and quite pos-sibly transform the polypeptide portion are clearlyproblems that both need to be solved, and should,in the course of their solution, throw much lighton the nature of lipoprotein structure.

The demonstration of partially delipidatedalpha lipoprotein (alpha LPB) in the ultracentri-fugal fraction of density > 1.21 is quite con-sistent with its accounting for some of the phos-pholipid long known to be in this fraction (19,39-41). Indeed this phospholipid has been shownby Kunkel and Trautman to migrate with thealpha1 globulin on starch block electrophoresis(41). The interesting suggestion has been madethat an apoprotein circulates in plasma, at a den-sity of > 1.21 that can be converted to a lipo-protein by the liver (42). A partially delipidatedlipoprotein such as alpha LPB could be identicalto such a carrier and this possibility will have tobe explored.

Summary

The homogeneity of alpha1 or high density lipo-protein (density 1.063 to 1.21) (HDL) has beenstudied by immunochemical techniques in combi-nation with electrophoresis, preparative and ana-lytical ultracentrifugation, and amino acid analy-sis. Lipoproteins from the following sources havebeen examined: fresh plasma, plasma aged for afew days at 40 C, or freeze-thawed repeatedly,delipidated, or exposed to 8 M urea, and ultra-centrifugal fractions including HDL (density1.063 to 1.21), HDL, (density 1.063 to 1.1),HDL3 (density 1.1 to 1.21), and the fraction ofdensity greater than 1.21.

With the exception of fresh plasma and HDL2,alpha lipoprotein from all the other sources wasreproducibly shown to exist in two forms. Thesehave been designated alpha LPA and LPB. Thesetwo forms have differing mobility on agar gelelectrophoresis, and 7 of 8 antisera prepared in

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three different animal species have found themto be only partially cross-reactive as antigens.

Only very small amounts of alpha LPB werefound in fresh plasma, and presumably alphaLPA is the native form in which these lipoproteinscirculate. Both forms contain protein of the sameamino acid composition and amino terminalgroups. Alpha LPB contains relatively less lipidand is judged to be of a greater average densityby its flotation characteristics. Alpha LPB wasshown to be formed from alpha LPA by a partialdelipidation.

It is concluded that repeated ultracentrifuga-tion of alpha LPA is always associated with itstransformation in part to alpha LPB. It was alsodemonstrated that the standard high densitylipoprotein fractions obtained by ultracentrifuga-tion are consistently related to, and their relativeconcentrations possibly explained by, this trans-formation. The HDL2 peak of density 1.063 to1.1 obtained by Gofman and colleagues in theanalytical ultracentrifuge consists solely of LPA,whereas the HDL3 peak (density 1.1 to 1.21)consists of alpha LPB plus some alpha LPA.Alpha LPB is also consistently demonstrated byimmunochemical techniques in the fraction of den-sity greater than 1.21, indicating that at leastafter ultracentrifugation, some alpha lipoproteinis present in a form of greater density than thatusually attributed to the lipoproteins.

The relationship of these findings to earlier dis-crepancies in studies involving the immunochem-istry or quantitation of high density lipoproteinsis discussed.

AcknowledgmentsWe thank Miss Nanci Briggs and Mr. Armando San-

doval for their technical assistance and Dr. John T. Pottsfor his helpful suggestions.

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heterogeneity of plasma high density lipoproteins€¦ · * all high density lipoprotein...

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