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EFFECTS OF HEPARIN ON THE LIPOPROTEINS IN HYPERLIPEMIA. ANELECTROPHORETICSTUDYOF THE SERUMALPHA ANDBETA

LIPOPROTEINS AFTER THEIR SEPARATIONBY FRACTION-ATION OF THE PLASMAPROTEINS OR ULTRACEN-

TRIFUGAL FLOTATION 1

By FRANZ S. M. HERBST, WALTERF. LEVER, MARYE. LYONS, ANDNANCYA. HURLEY

(From the Department of Dermatology, Harvard Medical School, and the Derinatologic Re-search Laboratory, Massachusetts Genteral Hospital, Boston, Mass.)

(Submitted for publication October 22, 1954; accepted November 17, 1954)

The effects of heparin on the electrophoreticpattern of serum have recently been studied in thisLaboratory. It had been found that in normal,strictly fasting individuals an intravenous injectionof heparin caused no changes in the patterns ob-tained either by moving boundary electrophoresisor by paper electrophoresis (1-4). On the otherhand, in alimentary hyperlipemia and idiopathichyperlipemia electrophoretic changes were ob-served. In the patterns obtained by movingboundary electrophoresis the changes consistedof a decrease in the beta-1 globulins to a sub-normal value and the appearance of a new com-ponent ahead of albumin (1, 2). On the basis ofthis observation it had been assumed that thepre-albumin component consisted of beta lipo-proteins. However, subsequent studies by paperelectrophoresis revealed that after an injection ofheparin not only the beta lipoproteins but also thealpha lipoproteins showed an acceleration of theirelectrophoretic migration: The beta lipoproteinlipid band moved with the speed of either alpha-2globulin, alpha-1 globulin, or intermediary be-tween alpha-1 globulin and albumin and the alphalipoprotein lipid band moved faster than albumin(3, 4).

Because the findings by paper electrophoresisindicated that the pre-albumin component con-sisted of the alpha lipoproteins and not of the beta-lipoproteins, as we had originally assumed, it wasthought worth while to study in greater detailthe effects of heparin on the alpha and beta lipo-

1 This investigation was supported by research grantsfrom the National Institute of Arthritis and MetabolicDiseases, of the National Institutes of Health, PublicHealth Service (PHS A-414), and from the MiltonFund, Harvard University.

proteins after their separation from one another.Consequently, electrophoretic analyses were car-ried out on plasma protein fractions and on thelipoproteins isolated by ultracentrifugal flotationin 10 normal fasting persons, two persons withalimentary hyperlipemia and seven patients withidiopathic hyperlipemia before as well as onehour after an intravenous injection of 100 mg. ofheparin.

METHODS

Collection of blood. Blood was withdrawn in a drysyringe, the samples were allowed to clot at room tem-perature, then centrifuged and the serum removed witha pipette. For the fractionation of the plasma proteins an-other blood sample was withdrawn in a syringe containingacid citrate-dextrose solution as an anticoagulant (5).

Electrophoresis. Paper electrophoresis was carried out,as described in our previous communications (3, 4), ac-cording to the technique of Grassmann and Hannig (6)with subsequent staining of the proteins with AmidoBlack 10B, and of the lipids with Sudan Black B (7, 8).For the quantitative determination of the relative amountsof protein the paper strips stained for protein were im-mersed for half an hour in a mixture of alpha brom-naphthalene and paraffin oil. This produced sufficientclearing of the paper strips so that they could be readin a densitometer. The diagram thus obtained wasanalyzed by planimetry. Quantitative determination ofthe relative amounts of lipids on the lipid stained paperstrips gave variable and unreliable results which there-fore are not reported.

Moving boundary electrophoresis was performed in aPerkin-Elmer Tiselius electrophoresis apparatus, Model38, as described in previous communications (3, 9).

Fractionation. Fractionation of the plasma proteinswas carried out according to Method 10 of Cohn and hisco-workers (10), modified for small amounts of plasmaby Lever and his co-workers (5). Two main fractionswere obtained, Fraction I + II + III, containing the betalipoproteins, and Fraction IV + V, containing the alphalipoproteins. Since Fraction I + II + III contained

581

FRANZ S. M. HERBST, WALTERF. LEVER, MARYE. LYONS, AND NANCYA. HURLEY

fibrinogen, which on analysis by paper electrophoresis in-teracts with the supporting medium, two drops of athrombin solution containing 200 N.I.H. units per ml.were added to the fraction, following which the fibrinclot was removed. Electrophoretic analysis was carriedout on the two main fractions within twenty-four hoursafter the fractionation of the plasma.

Preparative uiltracentrifugationt. Spinco Model L wasused as preparative ultracentrifuge. The rotor wasSpinco Model No. 40 in which the tubes are fixed at anangle of 26 degrees. Centrifugation was carried out at16 C. for approximately fifteen hours at a speed of35,000 R.P.M. which is equivalent to a gravitationalforce of 80,730 G.

For preparative ultracentrifugation at a solution den-sity of 1.063 Gm. per ml. 3 ml. of serum were mixedwith 7 ml. of a sodium chloride-water solution of suchdensity to give a final density of 1.063 Gm. per ml. at26 C. After centrifugation the uppermost 1 ml. contain-ing the low density lipoproteins, or beta lipoproteins, wasthen pipetted off (11).

For preparative ultracentrifugation at a solution den-sity of 1.21 Gm. per ml. 3 ml. of serum were mixed with7 ml. of a potassium bromide-water solution of suchdensity to give a final density of 1.21 Gm. per ml. at26 C. After centrifugation the uppermost 1 ml. waspipetted off. In it were present the low as well as thehigh density lipoproteins, i.e., the alpha and beta lipo-proteins (12, 13).

Paper electrophoresis of the isolated lipoproteins wascarried out within twenty-four hours after ultracentrifu-gation.

RESULTS

Plasma fractions

Fasting normial personis. Fraction IV + V onanalysis by paper electrophoresis and subsequentstaining for proteins contained, just as on analysisby moving boundary electrophoresis (10, 5), mostof the albumin of the serum, most of the alpha-1globulins, and small amounts of the alpha-2 andbeta' globulins (Figure 1). On the paper stripsstained for lipids one lipid band was present inthe alpha-1 globulin area representing the alphalipoproteins.

Fracticn I + II + III showed on the paperstrips stained for proteins, just as onimovingboundary electrophoresis, small amounts of albu-min, most of the alpha-2 and beta globulins andall the gammaglobulin (Figure 2). Paper stripsstained for lipids showed one lipid band in thebeta-1 globulin area representing the beta lipo-proteins.

Injection of heparin into 10 normal fasting per-sons caused no changes in the paper electropho-

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FI(.. 1. PAPER ELECTROPHORETIC PATTERNS OF THEAl.P11A LIPOPROTEIN FRACTION (FRACTION IV + V) OFA NORMAI INDIVIDUAL

On the lipid stained paper strip (L) one band is pres-eirt in the alpha-1 globulin area, representing the alphalipoproteins.

retic patterns of either Fraction IV + V or Frac-tion I + II + III.

Idiopathic hyperlipeniia. Analysis of FractionIV + V (alpha lipoprotein fraction) by paperelectrophoresis and subsequent staining for pro-teins and lipids in seven patients with idiopathichyperlipemia revealed a normal pattern. One

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FI(;. 2. PAPER ELECTROPHORETIC PATTERNS OF THEBETA LIPOPROTEIN FRACTION (FRACTION I + II + III)OF A NORMALINDIVIDUAI

On the lipid stained paper strip (L) one band is pres-ent in the beta globulin area, representing the beta lipo-proteins.

582

EFFECTS OF HEPARIN ON THE LIPOPROTEINS IN HYPERLIPEMIA

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PA AFIG. 3. PAPER ELECTROPHORETIC PATTERNS OF THE

ALPHA LIPOPROTEIN FRACTION (FRACTION IV + V) OFA PATIENT WITH IDIOPATHIc HYPERLIPEMIA AFTER ANINTRAVENOUSINJECTION OF 100 MG. OF HEPARIN

The alpha lipoprotein band on the lipid stained paperstrip (L) moves faster even than albumin as a pre-albu-min component (PA). The protein stained paper strip(P) shows that the pre-albumin component contains asmall amount of protein.

hour after the intravenous injection of 100 mg. ofheparin, staining for lipid revealed that the onelipid band present (alpha lipoprotein) had in-

creased its speed of migration and migrated aheadof albumin (Figure 3). The protein stainedpaper strip showed that this pre-albumin com-ponent contained small amounts of protein.Analysis of Fraction IV + V by moving bound-ary electrophoresis also showed the pre-albumincomponent (Figure 4).

Analysis of Fraction I + II + III (beta lipo-protein fraction) by paper electrophoresis andsubsequent staining for proteins revealed a greaterintensity of staining of the beta globulin bandthan in normal fasting persons. On staining forlipids the lipid band present in the beta globulinarea (beta lipoprotein) stained much more in-tensely than normal. Furthermore, just as in thepatterns obtained on whole serum (4), the betalipoprotein band showed a densely stained trailextending back to the starting line. This trailwas due mainly to the chylomicra. One hour af-ter the injection of heparin, the trail of the betalipoprotein band showed a considerable decreasein its staining intensity and the beta lipoproteinband itself moved with the speed of alpha-2 oreven alpha-1 globulin (Figure 5). The proteinstained paper strip showed a small amount of pro-tein moving with the same increased speed as thebeta lipoprotein on the lipid stained paper, repre-

PA A'4" PA.FIG. 4. PATTERNS OBTAINED BY MOVING BOUNDARYELECTROPHORESISIN A

PATIENT WITH IDIOPATHIC HYPERLIPEMIALeft, fasting serum: the alpha-2 peak is slightly and the beta-1 peak is moder-

ately elevated. Middle, serum after an intravenous injection of 100 mg. of heparin:the beta-i peak has decreased to a subnormal size and a new component has ap-peared ahead of albumin (PA). Right, alpha lipoprotein fraction (Fraction IV +V) after an intravenous injection of heparin: The pre-albumin component (PA) ispresent in this fraction.

583

584 FRANZ S. M. HERBST, WALTERF. LEVER, MARYE. LYONS, AND NANCYA. HURLEY

FIG. 5. PAPER ELECTROPHORETIC PATTERNS OF THEBETA LIPOPROTEIN FRACTION (FRACTION I + II + III)OF A PATIENT WITH IDIOPATHIC HYPERLIPEMIA AFTERAN INTRAVENOUSINJECTION OF 100 MG. OF HEPARIN

The beta lipoprotein band on the lipid stained paperstrip (L) moves with the speed of alpha-i globulin.

senting the protein moiety of the beta lipoproteins.Satisfactory patterns by the moving boundarymethod could not be obtained because of the milkyappearance of Fraction I + II + III, caused bythe presence of large amounts of chylomicra.

Isolated lipoproteinsFasting normal persons. Paper electrophoresis

carried out on the lipoproteins isolated by ultra-

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LIPOPROTEINS OF A NORMAL INDIVIDUAL ISOLATED BY

ULTRACENTRIFUGAL FLOTATION AT A DENSITY OF 1.21GM. PER ML.

Two bands representing the alpha and beta lipoproteins,respectively, are present on the lipid stained (L) as wellas on the protein stained (P) paper strip.

centrifugal flotation at a density of 1.21 Gm. perml. showed two bands on the lipid stained as wellas on the protein stained paper strip indicatingthe presence of both the alpha and beta lipopro-teins (Figure 6). Comparison of the lipid stainedpaper strip with the one stained for proteins con-firmed the fact, established already by chemicalanalysis (14), that the alpha lipoproteins have arelatively low lipid-protein ratio in contrast to thebeta lipoproteins which have a high lipid-proteinratio.

Paper electrophoresis of the lipoproteins iso-lated by flotation at a density of 1.063 Gm. perml. showed only one band on the protein andlipid stained paper strips representing the betalipoproteins (Figure 7). Again the relativelyhigh lipid-protein ratio of the beta lipoproteinswas apparent.

Injection of heparin into 10 fasting normalpersons did not cause any changes in the electro-phoretic patterns.

Idiopathic hyperlipemia. Analysis by paperelectrophoresis of the lipoproteins floated at adensity of 1.21 Gm. per ml. (alpha and beta lipo-proteins) revealed two distinct bands on boththe protein and lipid stained paper strips represent-ing the alpha and beta lipoproteins, respectively.The intensity of staining of the beta lipoproteinswas greater than normal on the protein stained

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FIG. 7. PAPER ELECTROPHORETIC PATTERNS OF THELIPOPROTEINS OF A NORMAL INDIVIDUAL ISOLATED BYULTRACENTRIFUGALFLOTATION AT A DENSITY OF 1.063GM. PER ML.

One band representing the beta lipoproteins is presenton the lipid stained (L) and on the protein stained (P)paper strips.


as well as on the lipid stained paper strip. As inthe analysis of whole serum and of Fraction I +II + III, the lipid stained paper strip but not theprotein stained paper strip showed considerabletrailing of the beta lipoprotein band, due to thechylomicra.

Analysis of the lipoproteins floated at a densityof 1.063 Gm. per ml. (beta lipoproteins) revealedthe same patterns as just described except that noalpha lipoprotein band was present.

An injection of heparin caused the followingchanges in the lipoproteins floated at a densityof 1.21 Gm.per ml. (alpha and beta lipoproteins):On the lipid stained paper strip the alpha lipo-protein band appeared much broader than beforeand the trail of the beta lipoprotein band repre-senting the chylomicra showed a decrease in itsstaining intensity. On the protein stained paperstrip three bands were now present instead of twobands (Figure 8). Comparison of the proteinstained with the lipid stained paper strip indicatedthat only the slowest moving of the three bandson the protein stained paper strip correspondedto the beta lipoproteins, whereas the two fastermoving bands corresponded to the alpha lipo-proteins. That the two faster moving bands rep-resented the alpha lipoproteins and only the third,slowest moving band represented the beta lipo-proteins was definitely proven by analysis of thebeta lipoproteins floated at a density of 1.063 Gm.per ml. since paper electrophoresis of the betalipoproteins thus obtained showed only one bandon the protein and lipid stained paper strips cor-responding to the slowest moving band.

In order to obtain information to which extentheparin had caused an increase in the speed ofelectrophoretic migration of the lipoproteins, bloodwas obtained before and after the injection of he-parin and the lipoproteins were isolated by flota-tion at a density of 1.21 Gm. per ml. They thenwere analyzed side by side in the same electro-phoresis chamber and both paper strips werestained for proteins. Comparison of the pre-heparin sample with the post-heparin sample re-vealed after the administration of heparin an in-crease in the electrophoretic mobility of the alphaas well as of the beta lipoproteins (Figure 8).The alpha lipoprotein band had split up into twobands of which the slower band showed only a

(3 ~~POST

FIG. 8. PAPER ELECTROPHORETICPATTERNS (STAINEDFOR PROTEINS) OF THE LiPOPROTEINS OF A PATIENT WITHIDIOPATHic HYPERLIPEMIA BEFORE (PRE) AND AFTER

(POST) AN INTRAVENOUS INJECTION OF HEPARINThe lipoproteins were isolated by ultracentrifugal flo-

tation at a density of 1.21 Gm. per ml. The pre-heparinand post-heparin samples were analyzed simultaneously.The alpha lipoproteins in the post-heparin sample are

split up into two components. Both alpha lipoproteinbands as well as the beta lipoprotein band show an in-crease in their electrophoretic mobility following the in-jection of heparin.

slight increase in mobility, was narrow and wellstained, while the faster band was broad andfaintly stained.

Alimentary hyperlipemia. Two normal personsin an alimentary hyperlipemic state were studied.They had received a meal consisting of 480 ml. ofheavy cream, 32 Gm. of sugar and 16 Gm. ofcocoa. Three and a half hours after the meal 100mg. of heparin were injected intravenously. An-alysis by paper electrophoresis of the plasma frac-tions and of the isolated lipoproteins before andafter heparin gave the same results as in idio-pathic hyperlipemia, except that the beta lipo-proteins on the protein-stained as well as on thelipid stained paper strip showed a normal stainingintensity.

Determination of the relative distribution of pro-teins between alpha and beta lipo proteinsThe alpha and beta lipoproteins of the serum

were isolated by flotation at a density of 1.21 Gm.per ml. before and after an injection of heparin.They then were analyzed by paper electrophoresisand the paper strips were stained for protein.

Determination of the relative amounts of pro-

585


tein present in the alpha and beta lipoproteinbands of the paper strips revealed in 10 fastingnormal individuals, on the average, 74 per cent ofthe proteins as alpha lipoproteins and 26 per centas beta lipoproteins (range of variation 24 to 27per cent). The average values in seven patientswith idiopathic hyperlipemia revealed a consider-ably higher percentage of proteins in the betalipoprotein band than in normal persons. namely42 instead of 26 per cent (range of variation 36to 51 per cent). This was indicative of the greatincrease in beta lipoproteins in this disease. Therelative values obtained in two normal individualsin an alimentary hyperlipemic state were the sameas in normal fasting persons.

Injection of heparin into 10 normal persons.seven patients with idiopathic hyperlipemia andtwo normal persons in an alimentary hyperlipemicstate caused no changes in the distribution of pro-teins between the alpha and beta lipoproteins inany of the cases.

DISCUSSION

For a study of the action of heparin on the lipo-proteins of normal and hyperlipemic sera twomethods were employed by us: Separation ofthe alpha lipoproteins froml the beta lipoproteinsby chemical fractionation, and isolation of the twolipoproteins by ultracentrifugal flotation at den-sities of 1.063 and 1.21 Gm. per ml. These twomethods in conjunction with analysis by paperelectrophoresis proved verx tuseful since they en-abled us to study the effects of heparin on thealpha and beta lipoproteins separately. The analy-ses by paper electrophoresis showed that frac-tionation of the plasma proteins by the small-scale modification of Cohn's Method 10 resultedin a clear-cut separation of the alpha lipoproteinsfrom the beta lipoproteins since each was presentin one fraction exclusively. Even after the injec-tion of heparin complete separation of the twolipoproteins was obtained.

Effects of heparin on the electrophoretic mobilityof the lipoproteins

Injection of heparin caused no change in theelectrophoretic mobility of the separated or iso-lated lipoproteins in normal individuals. On theother hand, in persons with either alimentary oridiopathic hyperlipemia both the alpha and beta

lipoproteins showed an increase in their electro-phoretic mobility. Of especial interest were thefindings on chemical fractionation where after theinjection of heparin the beta lipoproteins on thepaper strip used for the analysis of Fraction I +II + III miioved with the speed of alpha-2 oralpha-I globulin, while the alpha lipoproteins onthe paper strip used for the analysis of FractionIV + V" nmoved faster than albumin. This l)ro-v ided conclusive evidence that the pre-albumiincomponent which we had observed following theinjection of heparin in the electrophoretic patternsof whole serum in idiopathic hyperlipemia (1, 2)as well as in alimentary hyperlipemia (3) repre-sented the alpha lipoproteins.

Does heparin caufse a transforrnation of beta intoalpha lipoproteins?Several authors (15-19) have expressed the

opinion that heparin causes a transformation ofbeta lipoproteins into alpha lipoproteins. Boyle.Bragdon, and Brown (15), carrying out ultra-centrifugal analyses at a density of 1.21 Gm. perml., observed in alimentary hyperlipemia on in-jection of heparin and subsequent in vitro incuba-tion of the plasma an increase in the Sf classes rep-resenting the alpha lipoproteins. Nikkili (18),who also made in ziivo studies, found by analysiswith moving boundary and paper electrophoresisan increase in the amount of the alpha lipoproteinlipids in alimentarv hyperlipemia follow ing theinjection of heparin. This observation, however,merely indicates that the beta lipoproteins mi-grated with the speed of the alpha lipoproteinsand does not indicate an actual transformation ofbeta lipoproteins into alpha lipoproteins.2

2 The fact that Nikkila observed no pre-albumin com-ponent in alimentary hyperlipemia after the injection ofheparin may be due to the fact that he gave smalleramounts of cream (200 ml.) than we did (480 ml.) forthe purpose of inducing alimentary hyperlipemia andconsequently only the beta lipoproteins but not the alphalipoproteins showed an increase in the speed of electro-phoretic migration. This would be analogous to an ob-servation which we made on two individuals whom wehad presumed to be fasting but who had not been strictlyfasting (2). On moving boundary electrophoresis weobserved in both persons a decrease in the beta-1 globu-lin peak from normal to subnormal values together withan increase in the alpha-2 peak in one and in the alpha-Ipeak in the other. There was no pre-albumin peak pres-ent in either of these two persons.


The following are our reasons for believing thatan in vivo transformation of beta lipoproteins intoalpha lipoproteins by heparin does not occur:First, analysis of whole serum by paper electro-phoresis and subsequent staining for lipids nevershowed an increase in the staining intensity of thealpha lipoprotein band after the injection of hep-arin either in alimentary hyperlipemia (3) oridiopathic hyperlipemia (4). Second, as shownin the present study, isolation of the two lipo-proteins by ultracentrifugal flotation followed bypaper electrophoresis and staining for proteinsshowed after the injection of heparin neither inalimentary nor in idiopathic hyperlipemia anychange in the distribution of the proteins betweenalpha and beta lipoproteins. Third, chemicalfractionation of the plasma proteins by Cohn'sMethod 10 and determination of the amounts ofcholesterol and phospholipids present as alphalipoproteins and beta lipoproteins in idiopathichyperlipemia never revealed abnormally highamounts of lipids in the alpha lipoprotein frac-tion either after a single injection or after thelong-term administration of heparin; whereas theelevated values for cholesterol and phospholipidsin the beta lipoprotein fraction decreased (1, 20).Fourth, heparin was found to exert no effect onbeta lipoproteins with Sf values below 12 (21).Although beta lipoproteins with high Sf valueswere successively transformed into beta lipopro-teins with lower Sf values but slight changes onlyoccurred in those normal individuals whose serumcontained solely beta lipoproteins with Sf valuesof 20 and below; and no changes took place innormal individuals who had only beta lipoproteinswith Sf values below 12. Since the gradual trans-formation of the low density beta lipoproteins intothose of higher density stops after the beta lipo-proteins have incurred a certain increase in den-sity it seems unlikely that the beta lipoproteinscould change all the way into high density alphalipoproteins.

Cause of the increase in electrophoretic mobilityof the alpha and beta lipoproteins after heparin

According to our present knowledge heparinhas a direct effect only on beta lipoproteins ofhigh Sf values causing their transformation intothose of lower Sf values, but apparently has no

effect on beta lipoproteins of low Sf values and onthe alpha lipoproteins. How then does heparincause an increase in the electrophoretic mobilityof the alpha as well as of the beta lipoproteins inhyperlipemic sera? It appears likely that theseelectrophoretic changes are a secondary effect ofthe action of heparin on the beta lipoproteins withhigh Sf values. Shore, Nichols, and Freeman(22) observed that in vivo heparinized plasmacaused a release of fatty acids from beta lipo-proteins provided they were obtained from lipemicsera and thus possessed high Sf values. Further-more, Gordon (23) by the in vitro addition ofoleate to normal serum produced an increase inthe mobility of the alpha and beta lipoproteins.

If one assumes that the increase in the electro-phoretic mobility of the alpha and beta lipoproteinsafter an injection of heparin is due to the bindingof fatty acids by these lipoproteins, as suggestedby Gordon's study, one could explain not only theincreased mobility of both lipoprotein classes inpersons with hyperlipemia but also the absenceof electrophoretic changes in strictly fasting nor-mal persons. Fasting serum contains beta lipo-proteins of low Sf values only. Thus very smallamounts of fatty acids are available for release byheparin. On the other hand, hyperlipemic serumcontains large amounts of beta lipoproteins withhigh Sf values and accordingly' large amounts offatty acids (24). Some of these fatty acids arereleased after an injection of heparin and thenbound to the beta lipoproteins of low Sf values aswell as to the alpha lipoproteins, thus increasingthe electrophoretic mobility of these lipoproteins.

SUMMARY

1. The action of heparin on the serum alpha andbeta lipoproteins in idiopathic and alimentary hy-perlipemia was studied by electrophoretic analysesof these two lipoproteins after their separation bychemical fractionation as well as after their iso-lation by ultracentrifugal flotation at densities of1.063 and 1.21 Gm. per ml.

2. Electrophoretic analysis of the alpha andbeta lipoproteins after their chemical separationrevealed in both types of hyperlipemia as the re-sult of an intravenous injection of heparin an in-crease in the electrophoretic mobility of both lipo-proteins. The separated alpha lipoproteins moved

587


faster than albumin and the separated beta lipo-proteins moved with the speed of either alpha-2 or

alpha-1 globulin.3. Electrophoretic analysis of the alpha and

beta lipoproteins after their isolation by ultra-centrifugal flotation also revealed an increase intheir electrophoretic mobility following the injec-tion of heparin. The alpha lipoproteins were pres-

ent as two components in the post-heparin patternsobtained by paper electrophoresis.

4. These studies allow the conclusion that thepre-albumin component which we had observedfollowing the injection of heparin in the electro-phoretic patterns of whole serum in idiopathic as

well as alimentary hyperlipemia represents thealpha lipoproteins.

5. No evidence was found in this study as wellas in our previous studies to support the recentlyproposed concept that the administration of he-parin causes a transformation of beta lipoproteinsinto alpha lipoproteins.

6. The increase in the electrophoretic mobilityof the alpha and beta lipoproteins in hyperlipemiafollowing the administration of heparin may be ex-

plained by the lipolytic effect of heparin and theresultant binding of fatty acids by the alpha andbeta lipoproteins.

REFERENCES

1. Lever, W. F., Smith, P. A. J., and Hurley, N. A.,Effects of intravenous heparin on the plasma lipo-proteins in primary hypercholesteremic xanthoma-tosis and idiopathic hyperlipemia. Science 1953,118, 653.

2. Lever, W. F., Smith, P. A. J., and Hurley, N. A.,Idiopathic hyperlipemia and primary hypercho-lesteremic xanthomatosis. III. Effects of intra-venously administered heparin on the plasma pro-

teins and lipids. J. Invest Dermat, 1954, 22, 71.3. Herbst, F. S. M., and Hurley, N. A., Effects of hep-

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4. Herbst, F. S. M., Lever, W. F., and Hurley, N. A.,Idiopathic hyperlipemia and primary hypercho-lesteremic xanthomatosis. VI. Studies of the se-

rum proteins and lipoproteins by moving boundaryelectrophoresis and paper electrophoresis beforeand after administration of heparin. J. Invest.Dermat, In press.

5. Lever, W. F., Gurd, F. R. N., Uroma, E., Brown, R.K, Barnes, B. A., Schmid, K., and Schulz, E. L.,Chemical, clinical, and immunological studies on

the products of human plasma fractionation. XL.

Quantitative separation and determination of theprotein components in small amounts of normalhuman plasma. J. Clin. Invest., 1951, 30, 99.

6. Grassmann, W., and Hannig, K., Ein quantitativesVerfahren zur Analyse der Serumproteine durchPapierelektrophorese. Hoppe-Seyler's Ztschr. f.physiol. Chem., 1952, 290, 1.

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10. Cohn, E. J., Gurd, F. R. N., Surgenor, D. M., Bar-nes, B. A., Brown, R. K., Derouaux, G., Gillespie,J. M., Kahnt, F. W., Lever, W. F., Liu, C. H.,Mittelman, D., Mouton, R. F., Schmid, K., andUroma, E., A system for the separation of thecomponents of human blood: Quantitative proce-dures for the separation of the protein componentsof human plasma. J. Am. Chem. Soc., 1950, 72,465.

11. De Lalla, 0. F., and Gofman, J. W., Ultracentrifugalanalysis of serum lipoproteins in Methods of Bio-chemical Analysis, ed. by David Glick, New York,Interscience Pbl., 1954, vol. I, p. 459.

12. Gofman, J. W., Lipoprotein transformations in healthand disease. Educational Proc. Permanente Hos-pitals, 1952, 2, 174.

13. Green, A. A., Lewis, L. A., and Page, I. H., Amethod for the ultracentrifugal analysis of alphaand beta serum lipoproteins. Federation Proc.,1950, 10, 191.

14. Oncley, J. L., and Gurd, F. R N., The lipoproteinsof human plasma in Blood Cells and Plasma Pro-teins, ed. by James E. Tullis, New York, N. Y.,Academic Press, 1953, p. 337.

15. Boyle, E., Bragdon, J. H., and Brown, R. K., Roleof heparin in in vitro production of alpha-i lipo-proteins in human plasma. Proc. Soc. Exper. Biol.& Med., 1952, 81, 475.

16. Brown, R. K., Boyle, E., and Anfinsen, C. B., Theenzymatic transformation of lipoproteins. J. Biol.Chem., 1953, 204, 423.

17. Williams, G. R., Lipoprotein transformations underthe influence of heparin and Clostridium Welchiialpha-toxin. Biochim. et Biophys. Acta, 1954, 13,72.

18. Nikkila, E., Studies on the lipid-protein relationshipsin normal and pathological sera and the effect of


heparin on serum lipoproteins. Scandinav. J.ain. & Lab. Invest, 1953, Vol. 5, Suppl. 8.

19. Niklkili, E., and Grasbeck, R., Heparin in lipoidnephrosis. Observations of the effects on edema,proteinuria, serum proteins, lipids and lipoproteins.Acta med. Scandinav., 1954, 150, 39.

20. Lever, W. F., Herbst, F. S. M., and Hurley, N. A.,Idiopathic hyperlipemia and primary hypercho-lesteremic xanthomatosis. IV. Effect of prolongedadministration of heparin on serum lipids in idio-pathic hyperlipemia. Arch. Dermat., 1955, 71, 150.

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