Jownal of Clinical InvestigationVol. 44, No. 12, 1965

The Differentiation of Exogenous and Endogenous Hyper-lipemia by Paper Electrophoresis *

ROBERTS. LEES t ANDDONALDS. FREDRICKSON(From the Section on Molecular Disease, Laboratory of Metabolism, National Heart Institute,

Bethesda, Md.)

In the clinical study of hyperlipemia, it is usefulto know whether the excess plasma glycerides aredue to inadequate clearance of dietary fat ("fat-induced" hyperlipemia) or to release of glycerideby the liver at a rate exceeding capacity for re-moval ("endogenous" hyperlipemia). When theendogenous glyceride appears to accumulate in re-sponse to increases in dietary carbohydrate, theterm "carbohydrate-induced" hyperlipemia is oftenused (2).

The most direct way to make this distinction isto feed a high-carbohydrate diet containing prac-tically no fat for 1 to 4 weeks, followed by an iso-caloric diet in which fat represents at least 40% ofcalories and to obtain serial plasma glyceridemeasurements during both dietary periods (2, 3).Sometimes, fat induction can also be inferred fromthe presence of a normal plasma cholesterol con-centration when the glycerides are markedly in-creased or by the observation that alimentary par-ticles'! (chylomicrons) rise to the top of theplasma under appropriate conditions, leaving aclear infranatant layer. The preparative ultra-centrifuge can sometimes be used to acceleratethis separation. If hyperlipemia arises from bothexogenous and endogenous sources, however, noneof these tests may provide a decisive answer.

* Submitted for publication June 11, 1965; acceptedAugust 19, 1965.

Presented in part before the Thirty-seventh AnnualScientific Sessions of the American Heart Association inAtlantic City, N. J., October 23, 1964, and published inabstract form (1).

t Address requests for reprints to Dr. Robert S. Lees,National Heart Institute, Bethesda, Md. 20014.

1 The name "particles" has been applied (4) to lipo-proteins of size large enough (> 0.1 /A) to be seen withthe light microscope. These particles all have D < 1.006and Sf > 400; they therefore represent a subclass of "verylow density lipoproteins" as defined in the ultracentri-fuge (Table II).

Two more specific methods have recently beendeveloped for characterization of the glyceride-rich lipoproteins and particles that occur in hyper-lipemia. Starch-block electrophoresis has beenadapted to define three different peaks of "par-ticles" in plasma, two being associated withexogenous hyperlipemia and one representing en-dogenously synthesized glycerides (5, 6). Poly-vinylpyrrolidone (PVP) density gradient floc-culation has been devised to achieve the sameseparations (6, 7).

Earlier experience with electrophoresis on paperin buffer containing albumin (1, 8, 9) has sug-gested that the distinction between the two formsof hyperlipemia might be made by this simple andpractical technique. The behavior on paper of thevarious glyceride-rich lipoproteins and particles inplasma is here compared with the results obtainedby other methods, and the possible value of paperelectrophoresis in studies of hyperlipemia is ex-plored.

MethodsSubjects

Seventeen volunteers participated in studies of the ef-fects of dietary fat and carbohydrate on plasma lipo-proteins. They were defined as normal because theirparents and siblings had no history of metabolic disease,their glucose tolerance was normal, and while on a freediet their plasma cholesterol and glyceride concentrationswere within 2 SD of "normal" mean values for this labora-tory (3). Fifteen of these subjects were 17 to 21 yearsof age (nine male and six female), and two were males30 and 40 years of age. Patients with hyperlipoproteine-mia were also studied (Table I). Five had the rela-tively rare familial fat-induced hyperlipemia, the "Type I"syndrome as defined elsewhere (10). Eleven other pa-tients (Table I) represented at least two other types ofhyperlipemia.

Metabolic diets

All subjects were fed ordinary foods, in amounts nec-essary to maintain constant body weight, as one of threemajor diets: 1) a normal diet in which fat represented 40

1968

THE USE OF PAPER ELECTROPHORESISIN THE DIAGNOSIS OF HYPERLIPEMIA

to 45% of calories, carbohydrate 45 to 55%o of calories,and protein the remainder; 2) a high-carbohydrate dietcontaining 70 to 80%o of calories as carbohydrate, less than5 g fat, and the rest protein; 3) a high-fat diet contain-ing 60 to 85%o of calories as fat, 5 to 15%o of calories as

carbohydrate, and the rest as protein.

Single fat feedings

A single large fat meal was fed to some subj ects so

that the glyceride particles of alimentary origin might beisolated in quantity. This was usually 250 g of corn oil,emulsified with skim milk, or as a custard with egg white.Occasionally either 100 g of mixed fat as bacon and eggs

or 250 g of cocoa butter was fed. Blood was drawn 3 to12 hours after the meal, and the plasma was stored atroom temperature until used. For these and all otherblood samples, disodium ethylene diamine tetraacetic acid(EDTA), 1 mg per ml of blood, was used as antico-agulant.

Isolation of lipoproteins and particles

Electrophoresis. Paper electrophoresis was performedby a modification of the Durrum hanging-strip methodwith barbital buffer containing 1%o albumin (8). Starch-block electrophoresis was performed by the method ofKunkel and Slater (11), as modified by Bierman, Gordis,and Hamlin (5). After electrophoresis for 16 to 20 hoursat constant current at an initial potential drop of 6 v per

cm, the starch block was cut into half-inch segments.These were placed in test tubes to which 3 ml of 0.9%osaline in 0.001 MEDTAwas added. After shaking andcentrifugation at 1,000 X g for 15 minutes, the supernatantwas removed for further analysis. Particle recovery,

calculated as recovery of the original sample turbidity inthe starch-block eluates, was about 75%o.

Flocculation techniques. Flocculation in PVP densitygradients was carried out according to the method ofGordis (7).

Preparative ultracentrifugation. Ultracentrifugation was

performed at 15° C in the Spinco model L ultracentrifugewith the 40 and 40.3 rotors. Supernates were removed bytube slicing. Particle concentrates were prepared by cen-

trifugation of plasma or starch-block eluates at D 1.006for 3 X 106 g minutes (4); the packed particles were

resuspended by repeated passage through a no. 25 needle.These preparations inevitably contained some very lowdensity lipoproteins, since ultracentrifugal separations inthis density range are not perfect. Particle-free infra-nates were used for isolation of very low density lipo-proteins (see Table II) by further centrifugation at D1.006 for 16 hours at 100,000 X g (4). The preparationswere washed once by ultracentrifugation into overlying0.9% saline solution in 0.001 MEDTA.

Nomenclature. Various names have been applied tothe lipid-protein complexes in human plasma, dependingon the analytical system used. These operational defi-nitions are correlated in Table II with alpha, beta, andpre-beta lipoprotein and chylomicron bands defined by thepaper electrophoretic method.

TABLE I

Characteristics of the hyperlipemic patientsparticipating in the studies*

Postabsorptive

LipoproteinSubject Sex Age pattern Glyceride

years mg/100 mlJ.P. M 32 Type 1 2,014P.P. M 18 Type I 4,361L.W. M 21 Type I 4,345G.W.J. F 18 Type I 3,374G.S. F 2 Type I 5,272I.R. M 60 Type III 811E.A. M 44 Type III 480J.G. M 45 Type III 1,310F.S. F 54 Type III 600J.I. M 36 Type III 1,424E.G. F *64 Type IV 7,760S.L. F 62 Type IV 1,721S.P. M 58 Type IV 285E.M. F 51 Type IV 250G.G. M 23 Type IV >1,000J.Gr. M 1 Type IV 497

* The lipoprotein pattern, expressed in terms of aclassification developed for familial hyperlipoproteinemia(10), and glycerides were determined while the patientwas on an ad libitum diet. More detailed description ofmost of these patients appears elsewhere (3).

Determination of glyceride fatty acid composition.Lipoprotein bands from paper electrophoretic strips wereeluted for analysis of glyceride fatty acids as follows:Strips were run in replicate. One was stained with oilred 0, and areas were cut from one or two unstainedstrips corresponding to the lipoprotein bands revealedby staining. These were further subdivided into pieces ofapproximately 1 X 0.1 cm and shaken for 5 minutes in12.5 ml of chloroform-methanol (2:1) in a glass-stop-pered centrifuge tube. The phases were split by addi-tion of 2.5 ml of aqueous H2SO4, 1: 2,000. The chloro-form phase was evaporated to dryness, taken up in 50 IAIof chloroform, and applied to thin-layer plates coatedwith silica gel G.2 The chromatograms were developedwith petroleum ether: ethyl ether: acetic acid (85:15:1)for approximately 45 minutes and air dried. The plateswere sprayed with a solution of 2,7-dichlorofluorescein inethanol and the triglyceride spot scraped into a glass-stoppered tube and transesterified with sodium methoxidein absolute methanol for 30 minutes at 65° C (13).

Gas-liquid chromatography. Fatty acid methyl esterswere quantified by gas-liquid chromatography on 12-footethylene glycol succinate columns at 190° C with argonas carrier gas and a radium ionization detector. Thecomposition of a National Heart Institute Standard D asdetermined by this system agreed within 10% for the ma-jor and within 20% for the minor components of thismixture (14).

The corn oil and cocoa butter that were fed to inducealimentary particles to appear in plasma, and lipoprotein

2 Merck, Darmstadt, Germany.

1969

197.0 ROBERTS. LEES AND DONALDS. FREDRICKSON

TABLE II

Definitions used in various procedures for lipoprotein analysis

Paper electrophoresis (8)*(See Figure 1)

Ultracentrifugation (12)

Starch block electrophoresis(5, 6)

PVPt density gradientflocculation (6, 7)

Chylomicrons

Beta lipoproteinsPre-beta lipoproteins

Alpha lipoproteins

Very low density lipopro-teins

Low density lipoproteins

High density lipoproteins

Primary particles

Secondary particles

Endogenous particles

Primary particles

Secondary particles

Endogenous particles

Mobility: zero, all remains at theorigin.

Mobility: that of #,-globulin.Mobility: slightly greater than

beta lipoprotein.Mobility: between a,-globulin

and albumin.

Sf > 20 and D < 1.006.

Sf 0-20 and D 1.006-1.063;identical to beta lipoprotein.

D > 1.063, identical to alphalipoproteins.

Mobility: alpha2; found only afterfat ingestion.

Mobility: beta; found only afterfat ingestion.

Mobility: alpha2; found postpran-dially in plasma, unrelated tofat ingestion.

Particles that flocculate at the topof the density gradient tube.

Those that flocculate at the bottomof the tube.

Those that are distributed as ahazy flocculate throughout muchor all of the tube.

* Numbers in parentheses refer tot PVP = polyvinylpyrrolidone.

, pertinent references.

chylo , pre"s

_~~~~~~~~~~~~~~~~~~~~~......

a__ _

** . . :.: .. : . . :: .::: . .: .... ::: .:: ........ .............. ........ .. . .: .:. :::

.:.: ::: . . . :* : .. : . ..... ..

..

. mL.

FIG. 1. A DIAGRAMOF THE FOURBANDSTHAT MAYBE SEEN IN HUMANPLASMAON PAPER ELECTROPHORESISIN BARBI-TAL-ALBUMIN BUFFER (6). The anode is to the right.

THE USE OF PAPER ELECTROPHORESISIN THE DIAGNOSIS OF HYPERLIPEMIA

ORIGIN.

PRE-(d

DIET

*::.:. : ...

.:..:* i . .: . .:e .. . ... .... :.: .:: ... :::: :. . . ::: ..Xa ------+A__ _me' i :. ..sBSe,, ........ °. -. . a

.. ... .... .... : ..... .. . :. :.: .;..... :. : : . :. . . . .. . . . . .: . -. . ..... .. . . ... .

.: :0* . .: Be::: : :.: ::.i : : ........ :. .. . . ..:...: ....:: .. ....:::*:: :

.:.. : ;:.: : .: . .:*.:.: . :. : .. ::: .: : . : .:: .;*:: : . :: ..

*:: .::::. .. :: :: :.

: .: :.* . . .: : . . . .:NORMAL HIGH-FAT

TRIGLYCERIDE 1550

mg/I0o ml

CHOLESTEROLmg/loo mt

475

415 1000

475 285

FIG. 2. LIPOPROTEIN AND LIPID ANALYSES DURING METABOLIC STUDY OF

PATIENT J.G. The plasma lipoprotein patterns (reading from left to right)are those obtained after 2 weeks of normal diet, 3 weeks of high-fat diet, and1 and 3 weeks of high-carbohydrate (CHO) diet.

fractions isolated by other methods, were extracted bythe method of Folch, Lees, and Sloane Stanley (15); thechloroform extracts were treated as described for theextracts from paper electrophoretic strips.

Chemical analyses. Cholesterol was estimated with theautoanalyzer (16). Triglyceride was determined by themethod of Jagannathan (17).

ResultsOrigin of the glycerides in the pre-beta band

Plasma was obtained from 12 normal subjectsand from 7 patients with hyperlipemia after they

had fasted overnight. All had been fed the high-carbohydrate diet for periods of 3 to 21 days. Apre-beta band was now present in the plasma ofevery normal subject and was enhanced if presentearlier in the hyperlipemic patients. In none ofthe plasma samples was there a chylomicron band.This effect of the high-carbohydrate diet on thelipoprotein pattern of J.G. (Table I), a patientwith carbohydrate-induced hyperlipemia, is shownin Figure 2. After he had eaten a normal diet for2 weeks, his plasma had a milky appearance in the

TABLE III

The fatty acid composition of the glycerides of pre-beta lipoprotein*

Subject Diet 14:0 16:0 16:1 18:0 18:1 18:2

Normal B.W. High CHO 2.8 41.3 7.8 2.7 40.0 5.4Normal L.K. High CHO 2.2 34.7 9.5 1.5 42.6 9.5Normal S.M. High CHO 4.2 35.7 8.3 2.9 40.2 8.7Normal N.R. High CHO 3.4 46.5 7.6 4.3 34.3 3.9Carbohydrate-

induced hyper- High CHO 3.4 43.7 7.9 4.3 33.0 7.7lipemic F.S.

Carbohydrate-induced hyper- High CHO 2.0 38.8 4.7 3.9 44.8 5.8lipemic J.I.

* The acids between 14:0 and 18:2 were quantified, and each was expressed as a per cent of the total. The fattyacids are represented by chain length and number of double bonds as suggested by Dole and co-workers (18). CHO= carbohydrate.

3. G.NIH- 1965

HIGH CHO3 WEEKS

1630

375

...

.. ..

:::....... ... . ........ .. ... ....

:.::: :::

o ;.

- w- -i 1S11|1 E | "" F " I -.: : . :: :

.:....: :. :..::. : . . .. ::... ...: ... .: . .. :.

.: :::- : . : .. :.::.:: : :: ::... ::: :: . : . :.

:::A: .:: :;. .. :: .. .:::.::: - : : : . .:...... ... : :HloH CHO

| WER

1971

ROBERTS. LEES AND DONALDS. FREDRICKSON

PR'E016N

:..----:-::.-..;' ''I ' CE' ;. SE.' aft,........:

---DIET-- NORMALt FATAFR 10etFT 25gn ACE............. 2600 660....70.....22.W.~l T- AT"'60 70

CHOLESTEROL 2SOnmg/lO hil-175.-:- 170 18

FIG. 3. LIPOPROTEIN AND LIPID ANALYSES DURING METABOLIC STUDY OFPATIENT G.S. The plasma lipoprotein patterns (reading from left to right)are those obtained after 1 week of normal diet, 10 days of fat-free diet, 4days at 10 g of fat per day, and 10 days at 25 g per day.

postabsorptive state. There were dense beta andpre-beta bands but no chylomicron band (Figure2). After he had been fed the high-fat diet for 3weeks, his plasma became clear and the pre-betaband practically disappeared. Within 1 week af-ter he was changed to a high-carbohydrate diet,hyperlipemia reappeared with accumulation ofpre-beta lipoproteins, but again there were no de-tectable chylomicrons (Figure 2).

The triglycerides in the pre-beta lipoproteinsfrom four of the normal subjects and two of thepatients were isolated by preparative paper electro-phoresis. As shown in Table III, these glyceridescontained a preponderance of fatty acids consistentwith their having originated mainly from endoge-nous synthesis (2, 18).

Origin of the triglycerides in the chylomicron band.

One hundred g of mixed fat was fed to five nor-

mal subjects in whose plasma neither chylomicronnor pre-beta bands were seen in the fasting state.Blood was drawn 8 to 12 hours later and theplasma subjected to paper electrophoresis. Ineach case, a faint chylomicron band, but no pre-beta band, appeared. The effect of feeding fatwas much more dramatic in a patient with fat-

induced hyperlipemia (Figure 3). While thischild (G.S., Table I) was on a normal diet and inthe postabsorptive state, her plasma contained on

electrophoresis an extremely dense chylomicronband. The beta, pre-beta, and alpha lipoproteinbands were barely perceptible. The chylomicronband disappeared after she had been switched toa high-carbohydrate, virtually fat-free diet. Atthis time there now appeared a dense pre-betaband similar to that seen in other subjects on thehigh-carbohydrate diet. The beta and alpha lipo-protein bands were also more apparent. Fourdays after she was placed on a diet including only10 g of fat per day, chylomicrons were again pres-ent in her plasma in the postabsorptive state.After she had been on 25 g of fat per day for 10days, her plasma contained an excess of chylo-microns similar to that present when she ate a nor-

mal diet (Figure 3).The divergence of the glyceride fatty acid pat-

terns in the chylomicron and pre-beta bands was

demonstrated simultaneously in E.M. (Table I),whose plasma, when she was in the fasting state,contained a distinct pre-beta band. Three hoursafter she was fed 50 g of corn oil, her plasma glyc-erides had risen from 265 to 435 mg per 100 ml,

1972

TRIGLY

THE USE OF PAPER ELECTROPHORESISIN THE DIAGNOSIS OF HYPERLIPEMIA

CHYLOMICRONS

origin

=fatty acid content of fed fat

=fatty acid content of glycerides fromch ylomicrons band of paper electrophoretic strip

14:0 16:0 16:1 18 0 18 1 18:2

Fatty Acid

FIG. 4. THE FATTY ACID PATTERNOF CHYLOMICRONGLYCERIDES IN 3-HOURPOSTPRANDIAL PLASMAOF PATIENT E.M. Each fatty acid is represented bychain length and number of double bonds as suggested by Dole and co-

workers (18).

and there were both a chylomicron and a pre-betaband. These were eluted and the glyceride fattyacid compositions compared (Figures 4 and 5).The fatty acids in the chylomicrons (Figure 4)closely resembled those of the corn oil, whereas

701PRE-3

60

origin

@ 50

=fatty acid contera 40

= =fatty acid conte

. 30 Pre-r band of pa

20

0

those in the pre-beta band (Figure 5) had a highercontent of palmitic and oleic acids than did thechylomicron glycerides and a much lower contentof linoleic acid. This fatty acid pattern of pre-beta glycerides was comparable to that seen in the

nt of fed fat

ant of glycerides fromiper electrophoretic strip

Fatty Acid

FIG. 5. THE FATTY ACID PATTERN OF PRE-fi GLYCERIDES IN 3-HOUR POST-PRANDIAL PLASMA OF PATIENT E.M. The fatty acids are represented as inFigure 4.

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c

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._

72

c._

60-

50-

40-

1973

14:0 16:0 16:1 18.0 18:1 18:2

ROBERTS. LEES AND DONALDS. FREDRICKSON

six subjects shown in Table III, with the exceptionof a moderate increase in linoleic acid content de-rived from the fed fat. Similar appearance ofsome fed fat in endogenous lipoproteins has pre-viously been reported (18).

Correlation of bands wuith particles separated byother methods

The lipoproteins and particles separated by pa-per electrophoresis, starch block electrophoresis,and PVP flocculation were compared in severalways, particularly to avoid artifacts that mightarise during attempts to isolate particles by onetechnique and sequentially run them on anothersystem.

Sequential electrophoresis on starch and paper.In plasma samples from a normal subject fed fatand from a patient with fat-induced hyperlipemia(P.P., Table I), primary and secondary particleswere separately isolated on starch blocks. Thesewere resuspended in isotonic saline and appliedto paper for electrophoresis. Both the primaryand secondary particles remained at the origin onpaper. Plasma was obtained from another patientabnormally subject to fat induction while he wason the normal diet (J. P., Table I). The particles

=L

0.28

..0rqrn Secondory

O24t'

0.20 _

WX

0,12-

0.08

0-04-

n X lL i1

Albuminn

I I I I I IV I

2 4 6 8 to 12 14 16 18

TUBE NUMBER

FIG. 6. A COMPARISONOF STARCH-BLOCK AND PAPER

ELECTROPHORESISOF ULTRACENTRIFUGALLYISOLATED NOR-

MAL ALIMENTARY PARTICLES. The line drawing repre-sents the turbidity of particles eluted from half-inch seg-ments from a starch block. The inset shows the identical;sample run on paper electrophoresis and stained for lipidwith oil red 0.

in this plasma were distributed on starch in asingle broad peak that extended from the beta toalpha2 regions. In the PVP gradient tube, theturbid particles formed distinct top and bottomlayers, but some opalescence was also presentthroughout the tube.

The starch block was cut in narrow regions inthe beta and alpha2 zones. The eluate from thebeta zone, corresponding to secondary particles asdefined by Bierman and his associates (Table II),all remained at the origin in the position of chylo-microns. The eluate from the alpha, zone of thestarch block was separated into two bands on pa-per. One remained at the origin, and the secondran as a pre-beta band. These results are com-patible with the presence of both "primary" and"endogenous" particles in the alpha, region onstarch (6). The primary particles behaved aschylomicrons on paper; the endogenous materialmigrated to the pre-beta region.

Simultaneous electrophoresis and flocculation.Plasma samples obtained from four normal sub-jects not less than 9 to 12 hours after they hadeaten 100 g of mixed fat were also simultaneouslyseparated by the three methods. In all samples,only secondary particles were present as defined bythe starch-block (5) or PVP technique (7). Onpaper electrophoresis, each plasma sample con-tained a chylomicron band but no pre-beta band.Plasma samples from patients E.G. and I.R. (Ta-ble I), which contained opalescence only in the in-termediate zone in the PVP gradient tubes as de-scribed for endogenous particles (6), contained bypaper electrophoresis a pre-beta band but nochylomicrons.

Analysis of particles isolated by ultracentrifuga-tion. Plasma was obtained from two normal sub-jects 12 hours after they had eaten 250 g of cocoabutter. The particle fraction was isolated by ul-tracentrifugation. Human albumin was added toa final concentration of 4% to provide a markerfor measurement of mobility and the suspensionsubjected to simultaneous starch-block and paperelectrophoresis. Figure 6 illustrates that whereasboth primary and secondary particles were pres-ent in the starch block, only a dense chylomicronband appeared on paper.

Trailing of pre-beta lipoprotein. On paper elec-trophoresis of hyperlipemic plasma, a "trail" oflipid-staining material is frequently seen extend-

1974

v.

THE USE OF PAPER ELECTROPHORESISIN THE DIAGNOSIS OF HYPERLIPEMIA

ing from the pre-beta region to the origin. Thieffect is due to the presence of lipoproteins an(particles of very low density (8, 9, 19). In th4present studies, trailing was seen in some degreewhenever pre-beta lipoprotein was present, everin pure preparations of this material from patientson fat-free diets (Figures 2 and 3). To examinethe relationship of the size of pre-beta lipoproteinsto their tendency to trail, plasma was obtainedfrom J.G. after he had been on the fat-free dietfor 3 weeks (Figure 2). Lipoprotein fractionswere separated from this lactescent plasma in thepreparative ultracentrifuge, under conditions de-fined in the nomogram of Dole and Hamlin (4),to obtain classes of lipoproteins corresponding ap-proximately to Sf > 105, Sf 400 to 105, and Sf 20to 400. The preparations were not further washedafter the initial separation. On paper (Figure 7),the Sf > 105 fraction extended from the pre-betaregion to the origin as a light and uniformlystained area. The major fraction of endogenousparticles (Sf 400 to 105) and the Sf 20 to 400lipoprotein fraction each formed a distinct pre-beta band again with a faint trail extending uni-formly to the origin. The staining of the trailproduced by particles of Sf 400 to 105 was propor-tionately heavier. The tendency of the pre-betalipoproteins and particles to trail appears to be di-rectly related to their size and inversely relatedto their density.

Pure chylomicron preparations do not show anytendency to migrate away from the origin, but in-stead diffuse as a discrete band around it (Fig-ure 6). When there are sufficient chylomicronsto form a discrete peak or band in the starch orPVP separation, they can be seen as a discreteband around the origin even in the presence of thetrail that commonly follows pre-beta lipoproteins.An example of this can be seen in Figure 3 (stripidentified as on 10-g fat diet). When the plasmais extremely lipemic, a one- or twofold dilutionwith saline sharpens this distinction.

Discussion

These studies demonstrate that paper electro-phoresis in buffer containing albumin is capableof separating chylomicrons from the particles andvery low density lipoproteins in which glyceridesof mainly endogenous origin are transported.

Sf >105 Sf 40-105 Sf 20-400

FIG. 7. THE TRAILING OF PRE-BETA LIPOPROTEINS. Theelectrophoretic patterns are those of ultracentrifugalsubfractions of pre-beta lipoproteins obtained from pa-tient J.G. The Sf > 10' fraction, of which there is verylittle, trails from the pre-beta region to the origin as auniformly stained area. There is proportionately greatertrailing of the Sf 400 to 105 than of the Sf 20 to 400subfraction.

Chylomicrons do not migrate on paper electropho-resis but remain at the origin as a discrete band.The glycerides in this band have a fatty acid pat-tern closely resembling the ingested fat. Thechylomicron band includes both the primary andsecondary particles as defined by starch-block elec-trophoresis (5) or PVPgradient flocculation (7).

In contrast, endogenous particles as defined byusing starch or PVP (6) and very low densitylipoproteins as defined in the ultracentrifuge mi-grate to the pre-beta position on paper. The pre-beta band contains glycerides with a fatty acidpattern consistent with their predominantly en-dogenous origin. A correlation of the results ob-tained by paper electrophoresis with those of othertechniques is presented in Table IV.

The chemical or physical bases for the con-venient separation obtainable on paper, which oc-curs only in the presence of albumin in the buffer

1975

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ROBERTS. LEES AND DONALDS. FREDRICKSON

TABLE IV

The relationship of the chylomicron and pre-beta bands obtained by paper electrophoresisto other lipoprotein analytical methods

Location in Approximate SiBand on paper Migration in starch-block PVP density by ultracentri-

electrophoresis electrophoresis (5, 6) gradient (6, 7) fugation (8)

Chylomicron Alpha2 (primary particles) Top > 400

Chylomicron Beta (secondary particles) Bottom > 400

(Endogenous particles] Throughout > 400Pre-beta Alpha2 Very low density the tube

lipoproteins 20-400

(8), are not entirely known. The pre-beta lipo-proteins have been shown to contain both alphaand beta lipoproteins, or at least their characteris-tic proteins (20), and this probably accounts forthe intermediate mobility of these lipoproteins onpaper and starch. Any relationship between theprotein content of chylomicrons and their immo-bility on paper is difficult to assess. There is noagreement as to the nature of possible "chylomi-cron proteins" (4, 21, 22), but their total proteincontent is very small (21), giving a low ratio ofcharge to mass. It seems most probable that thechylomicrons remain at the origin on paper be-cause of this and because of their large size.

Paper electrophoresis can be used to distinguishonly two groups of glyceride-bearing particles incontrast to either starch or PVP, which segregatethree types. The consolidation of what on starchare separately isolated as primary and secondaryparticles, however, can be considered a practicalvalue of the paper technique, providing the sim-plest distinction between exogenous and endoge-nous glycerides. Although the earlier work sug-gested otherwise (5), Bierman and Strandnesshave recently found (23) that primary and sec-ondary particles represent different particulateforms of alimentary glycerides.

The first two patients whose responses to dietwere described in the Results offered a clear-cutdistinction between fat and carbohydrate inductionof hyperlipemia. In many patients, however, hy-perlipemia is exacerbated by both high-fat andhigh-carbohydrate diets. Both chylomicrons andendogenous particles may simultaneously collectin plasma even while such patients are on a nor-mal diet. The distinction between the two typesof particles is preserved even though there may be

trailing of endogenous glycerides in the regionwhere the chylomicrons are concentrated.

We have used the paper electrophoretic tech-nique for the diagnosis and follow-up of severalhundred patients with abnormal blood lipid con-centrations. These include more than 50 pa-tients in whom the results were correlated withdirect demonstration of fat or carbohydrate induc-tion of hyperlipemia under carefully controlledmetabolic diets. Dietary testing and plasma glyc-eride analyses are still necessary to establishwhether endogenous hyperlipemia is due to ab-normal carbohydrate induction, which is onlyquantitatively different from the normal responseto increased dietary carbohydrate (24). How-ever, the paper technique does permit the recog-nition of fat-induced and "mixed" hyperlipemiawithout dietary testing and further enables one toestimate, at any point in time, the proportion ofplasma glycerides that has come from endogenousand exogenous sources. It provides a simple andrapid way to follow and record studies of lipo-protein metabolism. The lipoprotein patterns pro-vided by this paper electrophoretic system havebeen proposed as the basis of a new classificationof familial hyperlipoproteinemia (10).

Summary

The plasma lipoproteins and "particles" sepa-rated by paper electrophoresis in barbital buffercontaining 1 % albumin have been compared withthose separated by starch electrophoresis, poly-vinylpyrrolidone gradient flocculation, and theultracentrifuge. The lipid-protein complexes uti-lized were obtained from both normal subjects andpatients with hyperlipoproteinemia who were feddiets high in fat or in carbohydrate to produce an

1976

THE USE OF PAPER ELECTROPHORESISIN THE DIAGNOSIS OF HYPERLIPEMIA

increase in plasma glycerides of exogenous or en-

dogenous origin, respectively. The fatty acid con-

tent of these glycerides was analyzed after elutionfrom the paper.

Chylomicrons, which contain exogenous or ali-mentary glycerides, remain at the origin on paper

and correspond to both primary and secondaryparticles as defined by starch and polyvinylpyr-rolidone. Particles and very low density lipopro-teins (D < 1.006) containing endogenously syn-

thesized glycerides migrate to the pre-beta posi-tion on paper.

This simple system makes possible the immedi-ate recognition of fat-induced and "mixed" hyper-lipemia; it is a useful adjunct to dietary testing inthe establishment of carbohydrate induction ofhyperlipemia. It also provides, at minimal ex-

penditure, a useful means to follow and recordstudies of lipoprotein metabolism.

Acknowledgment

We gratefully acknowledge the technical assistance ofMiss Eleanor Gundling.

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