Separation of plasma lipoproteins by density-gradient ultracentrifugation

ANALYTICAL

BIOCHEMISTRY

Separation

65, 42-49 (1975)

of Plasma

Density-Gradient T. G. REDGRAVE,

Lipoproteins

by

Ultracentrifugation D. C. K. ROBERTS,

AND C. E. WEST Parkville.

Depurtment of Physiology, University of Melbourne. Victoria, 3052 (TGR) and Department of Experimental John Curtin School of Medical Research, Australian Natiorlal University. Carlberra City, A.C.T., 2601, Australk

Pathology,

Received April 15, 1974; accepted October 22, 1974 The separation of plasma lipoproteins into VLDL, LDL. and HDL by a single 24-hr ultracentrifugation in swinging-bucket rotors is described. The mean recovery of loaded lipoprotein cholesterol is 89.3 2 1.3%. The method employs a discontinuous salt gradient and separates VLDL, LDL. and HDL as verified by cellulose acetate- and immuno-electrophoresis. The method offers some advantages for research applications.

The original methods for separation of the different plasma lipoproteins have employed multiple centrifugations in angle-head rotors with adjustments of the density of the medium between centrifugations (l-3). As pointed out by Hatch and Lees (4) angle-head rotors have the disadvantages of convective disturbance and adherence of large lipoprotein molecules to the walls of the centrifuge tube. These effects are much reduced in swinging-bucket rotors which have, therefore, been used to subfractionate chylomicrons (5,6), VLDL’ (6-8), LDL, and HDL (7,8). The lipoprotein species have thus been divided into several subfractions by these cumulative rate-centrifugation techniques. Experience is now reported of a simple discontinuous density-gradient procedure using high-speed swinging-bucket rotors to separate the individual plasma lipoprotein species in a single ultracentrifugation. The method provides a useful means of studying lipoprotein metabolism on relatively small samples in experimental situations and perhaps could be applied to the routine analysis of plasma lipoproteins. I Abbreviations: very low-density lipoprotein, VLDL, low-density high-density lipoprotein, HDL. very high-density lipoprotein, VHDL, tetra-acetic acid. EDTA. 42 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

lipoprotein. LDL, ethylene diamine-

PLASMA

LIPOPROTEIN

MATERIALS

ULTRACENTRIFUGATION

AND

43

METHODS

Blood was collected from subjects into tubes containing EDTA (final concentration 4 mM, pH 7.4) either at 9.0 AM after an overnight fast or before the midday meal in unfasted subjects. Plasma was separated by centrifugation at 300g for 15 min at 4°C. No attempt was made to remove chylomicrons from the midday samples because the results were not obviously different from the samples taken at 9.0 AM. Lipoprotein samples were usually prepared on the same day or within 72 hr of collection. Plasma cholesterol concentration was determined in these samples prior to ultracentrifugation. An aliquot was saponified by the method of Mann (9) and the cholesterol was extracted into petroleum ether (bp 60-80°C) and assayed using the o-phthaldialdehyde color reagent of Zlatkis and Zak (10). Three types of 6 X 14-ml titanium swinging-bucket rotors have been used in their respective centrifuges: the SW 41 rotor in the Beckman L3-50 ultracentrifuge (Beckman Inc., Palo Alto, CA 94034), the M.S.E. rotor (Cat. No. 43 127- 111) in the M.S.E. Superspeed 65 Mk II ultracentrifuge (Measuring and Scientific Equipment Ltd., London S.W.1, U.K.), and the SB-283 rotor in the I.E.C. B-60 ultracentrifuge (International Equipment Co., Needham Heights, MA 02194). Samples of plasma were adjusted to d = 1.2 1 with solid potassium bromide (0.325 g/ml plasma) and 4.0-ml aliquots were pipetted into 13.5 ml cellulose nitrate centrifuge tubes (9/16-in. diam X 3%in. length). Samples of less than 4.0 ml were adjusted with salt solutions and solid potassium bromide to d = 1.21 in a volume of 4.0 ml. A discontinuous gradient was formed by carefully layering 3.0 ml of salt solution d = 1.063 above the plasma, followed by 3.0 ml of salt solution d = 1.019. Finally, the tube was filled with 2.5-3.0 ml of d = 1.006 salt solution. To minimize mixing at the density junctions, the salt solutions were allowed to gravity feed down the side of the centrifuge tube through a hypodermic needle (22 gauge) attached to a glass syringe with the barrel removed. All salt solutions contained EDTA (0.1 mg/ml) and were prepared from potassium bromide and sodium chloride (2). The samples were centrifuged for 24 hr at 20°C at either 4 1,000 rpm (SW 41, 386,OOOg: SB-283, 283,2OOg), or at 40,000 rpm (M.S.E., 284,000g). After centrifugation chylomicrons and VLDL were present at the top of the tube, LDL at the junctions of the next two steps of the density gradient, and high density lipoprotein (HDL) at the d = 1.2 1 and d = 1.063 junction. In human samples LDL was clearly visible as an orange-yellow band (Fig. 1). The density of the fractions after centrifugation was measured with a digital precision density meter (Mode1 DMA 02C, Anton Pear KG, A-8054 Graz, Austria).

44

REDGRAVE,

ROBERTS

FIG. 1. Photograph of gradient in centrifuge lipoproteins. On the left are marked the fractions Marks on the centrifuge tube indicate the points band III LDL is clearly visible as a distinct samples.

AND

WEST

tube after separation of human plasma of the gradient that comprise bands I-IV. of discontinuity in the original gradient. In band, colored orange-yellow in human

The bands, together with part of the salt solutions above and below each band were carefully transferred by aspiration into tubes, saponified, and assayed for cholesterol as described. In some cases an aliquot was removed before saponification and dialyzed overnight at 4°C against salt solution d = I.019 prior to immunoelectrophoresis (11). Electrophoresis in agar using LKB equipment (LKB Produktor A.B., Stockholm 12, Sweden) separated the proteins of the dialyzed bands. The purity of the bands was checked with anti-sera to a,-lipoprotein, P-lipoprotein, and whole human serum purchased from Behringwerke A.G., Marburg-Lahn, Germany. The precipitin lines were stained sequentially for lipid using oil red 0 (0.5% w/v) in ethanol (50% v/v) and for protein using light-green SF (05% w/v) in t~chloroacetic acid (5% w/v). RESULTS

Table 1 shows the distribution of lipoprotein cholesterol in 19 normal male subjects. Total recovery of loaded lipoprotein cholesterol is 89.3 rt 1.31% @EM). In these subjects the plasma cholesterol concentrations range from 135 to 287 mgldl, with a mean of 2 13 & 9.6 (SEM)

PLASMA

LIPOPROTEIN

TABLE DISTRIBUTION

OF LIPOPROTEIN

45

ULTRACENTRIFUGATION 1

CHOLESTEROL

IN

19

NORMAL

MALE

SUBJECTS

Cholesterol distribution” Band

Density

Designation

% of total

I

1.006

7.3 ? 0.81

II III IV -

1.006-1.019 1.019-1.063 1.063-1.21 >1.21

VLDL and chylomicrons LDL LDL HDL

11.4 50.1 24.2 1.0

k f f i-

mg/dl 15.4 k 1.78 23.9 109.2 49.8 14.7

2.22 2.52 1.68 0.57

+ k 2 2

4.36 8.78 3.01 1.17

a Figures are mean *SEM.

mg/dl. As expected, most of the plasma cholesterol is recovered in the bands corresponding to LDL (bands II + III = 61.5 k 2.01%), with the majority in band III at 1.019 < d < 1.063. In band IV (1.063 < d < 1.21), 24.2 2 1.68% is recovered, corresponding to HDL. A further 7.0 2 0.57% is recovered in the fraction d > 1.21. During the course of the centrifugal procedure there is a smoothing of the discontinuous gradient as shown in Fig. 2. However, the general 1240Too of lube 1.210 .

2 Gi c?

1063 .

1019 . 1006 ,

1

4

3

5

7 VOLUME

9

11

13

(ml)

2. The shape of the gradient after centrifugation. Measurements were made with a digital precision density meter. The shape of the gradient when initially formed is shown by the dotted line. It should be noted that the ordinate is a logarithmic scale. FIG.

46

REDGRAVE,

ROBERTS

AND

TABLE PRESENCE

OF PRECIPITIN BANDS

LINES AFTER

I I1 III IV d > 1.21 Whole serum

2

TO SPECIFIC

of lipid-staining various

Anti-whole (1) (1) 1 1 1 Many

a See Table 1 for densities and designations * ( ) indicate the presence of a weak line. e Antisera to o(i- and P-lipoproteins.

ANTISERA

IN LIPOPROTEIN

IMMUNOELECTROPHORESIS

No.

Band”

WEST

precipitation antisera*

lines

to

Anti*,”

Anti-P

0 0 0 1 1 I

(1) (1) 1 0 0 I

of bands.

HS aH IV

FIG. 3. Immunoelectrophoresis of human lipoproteins isolated by ultracentrifugation. The bands were dialyzed at 4°C overnight against saline d = 1.O 19. Electrophoresis was carried out on aliquots of the bands at 2.50 V for I hr using LKB apparatus. Verona1 (bar-

PLASMA

LIPOPROTEIN

47

ULTRACENTR~FUGATION

200-

_

160-

K E" - 120?j ; cn 2

E30-

.I! " ii .a

40100

150 plasma

200 cholesterol

250

300

(w/d11

FIG. 4. Regression of LDL cholesterol on total plasma cholesterol. The equation of the line is y = 0.856.~ - 49.29. where .v = LDL cholesterol mg/dl and x = total plasma cholesterol mg/dl.

shape of the gradient is maintained, and the designated fractions are recovered where anticipated as shown in Fig. I. The technique could be adapted if necessary to a more extensive subfractionation of plasma lipoproteins because of this smoothing of the gradient during the centrifugation. With electrophoresis on cellulose acetate, band III and band IV travel as single bands corresponding to LDL and HDL. Bands I and II run as diffuse bands from the origin to the pre-P region. Immunoelectrophoresis of the bands in agar show there is no cross contamination of LDL and HDL and that each band is free of other plasma proteins (Table 2). Band IV shows only a single lipid-staining precipitin line with anti-a,lipoprotein serum and with anti-human serum. Similariy band III shows only a single precipitin line with anti-@-lipoprotein serum and with antihuman serum (Fig. 3). Anti-&lipoprotein serum shows a weak precipitin line with bands I and II but anti-~*-lipoprotein serum does not react. The band of d > 1.21 shows a single lipid-staining precipitin line with anti-~,-lipoprotein serum and with anti-human serum (Table 2) indicating the presence of some residual HDL in this fraction (see discussion). Figure 4 shows the correlation between total plasma cholesterol and LDL cholesterol concentrations. The correlation coefficient between the bital) buffer pH 8.6, ionic strength 0.1 was used. aH = anti-whole human serum: aB = anti/3-lipoprotein serum: Hs = whole human plasma: III = band III from ultracentrifugation: IV = band fV from uftracentrifugation. See Table I for densities and designations of bands.

48

REDGRAVE,

ROBERTS

AND

two parameters is 0.93 and the equation y = 0.856 x - 49.29.

WEST

of the regression

line is

DISCUSSION

The reported method provides a convenient and rapid ultracentrifugal analysis of the plasma lipoproteins. The discontinuous gradient is simple to prepare and inherently stable if mechanical disturbance is carefully avoided. Separation is dependent only on the densities of the lipoprotein species so separations are quite distinct. From the relationship shown by Dole and Hamlin (12) for flotation of lipoprotein species, calculations show that 24-hr centrifugation is sufficient for quantitative recovery of all VLDL and LDL of density 1.006-1.019 in the correct region of the gradient. Calculations show that 41-hr centrifugation is required for quantitative recovery of LDL of density 1.O19- 1.063, and 55 hr for all of the HDL to migrate to the top of the density = 1.21 band. In practice, however, a 24-hr centrifugation yields satisfactory separation of these components as shown by immunoelectrophoresis. Analytical results after a 40-hr centrifugation of rabbit plasma are unchanged. Presumably the bulk of these lipoprotein classes has either a larger mean diameter or is less dense than the limiting conditions of the calculations. The recovery of 7% of cholesterol in the fraction d > 1.2 1 is in keeping with the findings of other workers (13,14). It may be explained by the presence of VHDL, or by artifactual dissociation of lipoproteins during the process of ultracentrifugation (14). The subfractionation of LDL into two bands in this method requires further comment. Band II corresponds to the LDL I + LDL II subfractions of Lee and Alaupovic (15), whereas band III of the present method corresponds with the LDL III-LDL VI subfractions (15). Fundamental differences exist between these two groups of LDL subfractions (15) so their separation in this method is useful in assessing the metabolic significance of the LDL subgroups. We observed considerable variation in the amount of LDL in band II as illustrated by the relatively high standard error of the mean in Table 1. Because both chylomicrons and VLDL have density < 1.006 the described method does not separate these species. Chylomicrons can be separated from VLDL only by a preliminary rate-centrifugation method, for example as described by Minari and Zilversmit (16) and Hatch ef al. (5). By definition, and because of their very brief circulating half-life (17), chylomicrons will not be present in normal fasting plasma, and band I will consist entirely of VLDL. As expected from the fact that the major cholesterol-carrying fraction in normocholesterolemic human subjects is LDL, there is a close correlation between LDL cholesterol and total plasma cholesterol concentra-

PLASMA

LIPOPROTEIN

49

ULTRACENTRIFUGATION

tions. This is expected from the relationship previously shown by Havel, Eder, and Bragdon (2) and by Masarei and co-workers (18). In our laboratories, this ultracentrifugation method is being used in the analysis of plasma from cholesterol-fed rabbits with hypercholesterolemia. Such rabbits have increased amounts of cholesterol in VLDL and in LDL fraction II (19). The method is also being used to investigate the rapid metabolic interconversions and exchanges between lipoprotein classes. Hypercholesterolemic samples have been analyzed with satisfactory results, but further work will be needed before the routine application of the method to abnormal human samples. ACKNOWLEDGMENTS The research programs of one of us (TCR) are supported by grants from the National Heart Foundation of Australia and the National Health and Medical Research Council.

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7. Hinton,

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7,

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Mann. G. V. (1961) Clin. Chem. 7, 275-284. Zlatkis, A., and Zak, B. (1969) Anal. Biochem. 29, 143-148. Uriel, J., and Grabar, P. (1956) Ann. Inst. Pasteur 90, 427-440. Dole. V. P., and Hamlin, J. T. (1962) Physiol. Rev. 42, 674-701. Switzer, S., and Eder, H. A. (1965) J. Lipid Res. 6, 506-51 1. Levy, R. I.. and Fredrickson, D. S. (1965) J. Clin. Invest. 44, 426-441. Lee. D. M.. and Alaupovic, P. (1974) Biochem. J. 137, 155- 167. Minari. O., and Zilversmit, D. B. (1963) J. Lipid Res. 4, 424-436. Harris, K. L.. and Felts, J. M. (1970) J. Lipid Res. 11, 75-81. Masarei, J. R., Summers, M., Curnow, D. H., Cullen. K. J., McCall, M. G., Stenhouse, N. S.. and Welborn, T. A. (1971) Brit. Med. J. 1, 78-82. 19. Roberts, D. C. K., West, C. E., and Smith. J. B. (1973) Proc. Ausr. Biochem. Sot. 6, 9.

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