A novel method for the rapid separation of plasma lipoproteins using self-generating gradients of iodixanol

atherosclerosis Atherosclerosis 124 (1996) 125-135

Rapid communication

A novel method for the rapid separation of plasma lipoproteins using self-generating gradients of iodixanol J.M. Graham”, Joan A. Higgins b,*, T . Gillott’, T. Taylor’, Jane Wilkinsonb, Terry Ford”, David Billington” “School of Biomolecular Sciences, Liverpool John Moores University, Liverpool L3 3AF, UK bDepartment of Molecular Biology and Biotechnology, University of’ Shefield, Firth Court, Shefield SlOZTN, UK “Cardiology Laboratory, Northern General Hospital, Shefield, UK

Accepted 4 January 1996

Abstract We describe a new method for the rapid fractionation of plasma lipoproteins, which makes use of a new non-ionic, iodinated, density gradient medium, iodixanol, commercially available as Optiprep TM.The method is simple: plasma or serum is mixed with iodixanol followed by centrifugation in a vertical or near vertical rotor. Separation of VLDL, LDL and HDL can be achieved in 3 h and the lipoprotein fractions are comparable in density and composition with those prepared using conventional salt based gradients. Each class of lipoprotein can be removed in a single fraction, or a profile of lipoprotein distribution can be obtained using a gradient fractionator. Because the medium is inert, fractions from the gradient can be analysed by agarose gel electrophoresis or assayed for lipid content or apolipoprotein composition by SDS-PAGE without removing the iodixanol. Small differences in electrophoretic mobility of HDL and LDL across several gradient fractions suggest that subfractionation of these classesmay occur. The new method is simple, rapid and versatile with potential application for preparation of lipoproteins and for analysis of lipoprotein profiles in the research or clinical laboratory.

1. Introduction Routinely, fractionation of plasma lipoproteins (chylomicrons, VLDL, LDL and HDL) by centrifugation is carried out by flotation, either in a series of steps in which the density of the plasma is raised sequentially by addition of potassium * Corresponding author, Tel.: + 44 114 2824235;Fax: + 44 114 2728697.

bromide or through a discontinuous or continuous gradient of potassium bromide [l-5]. Both methods require prolonged centrifugation of up to 78 h for sequential flotation or at least 24 h for separation on discontinuous gradients. The use of short path-length vertical rotors [6] combined with collection and continuous analysis of the gradient by an autoanalyser has provided a rapid method ( < 60 min centrifugation) for analysis of the cholesterol content of lipoprotein fractions [7].

0021-9150/96/$15.000 1996 Elsevier Science Ireland Ltd. All rights reserved PII SO021 -9150(96)05797-g

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Such methods, however, separate only small volumes of plasma ( < 2 ml) and are analytical rather than preparative. For preparation and concentration of lipoproteins from larger volumes of plasma it is necessary to use a method involving longer centrifugation and for many applications this must be followed by overnight dialysis to remove the salts used in the gradient. Moreover, high ionic strengths can lead to elution of apolipoproteins and may interfere with the water structure around proteins leading to denaturation

PI.

In the present paper we report a new method for separation and concentration of plasma lipoproteins on self-generating gradients of iodixanol. This new medium forms solutions which can be made iso-osmotic at all densities and can form self-generated gradients in l-3 h [9]. Iodixanol is widely used as an imaging agent for injection into humans and it has therefore undergone rigorous trials and clinical screening [lO,l 11. It has also been shown to be inert, non-toxic to cells, and non-inhibitory to enzymes [9]. Thus, separated lipoprotein fractions can be analysed for lipid content, for apolipoprotein composition by SDSPAGE or separated by agarose gel electrophoresis without the extensive dialysis necessary for lipoproteins prepared on salt gradients. The separation of plasma lipoproteins on self-generated gradients of iodixanol is simple, rapid and versatile and can be used as a preparative or analytical technique. In this paper we report the principle and basic protocol of the method which can be readily adapted for different applications. 2. Materials and methods 2.1. Materials

OptiprepTM (60% w/v iodixanol) was a kind gift from Nycomed Pharma AS, Oslo, Norway. Maxidens and Nunc 96-well plates were obtained from Gibco. Hydrogel lipo + Lp(a) agarose gel kits were obtained from YSI Clandon. Monoclonal anti-human-apo-B (MAC131) was a kind gift from Dr. Ermanno Gherardi [12]. Anti-h-apo-Al, and cholesterol and triacylglycerol assay kits and

standards were obtained from Boehringer. Beckman Instruments (UK) Ltd., generously provided the following equipment: an Optima TLX (tabletop) ultracentrifuge with a TLNlOO near vertical rotor and 3.9 ml Quick-sealTMtubes for this rotor and a VTi 65.1 vertical rotor for the floor standing Beckman L7 or L8-80 ultracentrifuges. OptisealTM(11.2 ml) and g-maxTM(6.3 ml) centrifuge tubes were used in this rotor. Electrophoresis and immunoblotting reagents were as described previously [13] and all other chemicals were analytical grade. Blood samples were taken by venepuncture from patients routinely attending the Lipid Clinic of the Cardiothoracic Laboratory, Northern General Hospital, Sheffield and from healthy volunteers and serum was prepared by centrifugation (1000 g for 20 min). In some experiments blood was taken into 1 mM disodium EDTA as anti-coagulant and red blood cells pelleted by centrifugation (1000 g for 20 min). Serum or plasma sampleswere used fresh or were stored at 4°C and used within 24 h. The use of either plasma or serum had no influence on the subsequent gradient fractionation of lipoproteins. 2.2. Fractionation of lipoproteins Fifty percent (w/v) iodixanol solution prepared by diluting 5 vol of OptiprepTM with 1 vol of 0.8% (w/v) NaCl, 60 mM HEPES-NaOH, pH 7.4 (buffered saline) was used in several different protocols. Methods 1 and 2 were for small volumes of serum; Method 3 was for a larger volume of serum and in Method 4 a gradient was generated from two layers of iodixanol in which the serum occupied one or both of the layers. 2.2.1. Method 1 Serum (3 ml) was mixed with 50% iodixanol (1 ml) to give a final concentration of iodixanol of 12.5% (w/v). 3.5 ml of the mixture was transferred to Quick-seal tubes (volume 3.9 ml) for the TLNlOO near vertical rotor and the tubes were filled by layering buffered saline on top (about 0.5 ml). The tubes were sealed and centrifuged for 3 h in the Optima TLX bench top ultracentrifuge at 353 000 g,, (100 000 rpm) for 3 h at 16°C using

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triacylglycerol

cholesterol -I I30 total1

l-

2 3 4 5 6 7 8 9 1011 12 13 14 Fraction Number

Fig. 1. Separation of serum lipoproteins on iodixanol gradients. Human serum was separated on iodixanol gradients (starting concentration 12.5% w/v) in the TNLlOO rotor as described in Method 1. Fractions were collected from the bottom of the tube (fraction 14 is the top of the gradient) and 3 pl aliquots of the total serum and each fraction were separated on Hydrogel lipo + Lp-a agarose plates and stained with Sudan Black as described in Section 2 (top panel). Triacylglycerol (middle panel) and cholesterol (bottom panel) were determined directly on 2 ,ul aliquots of the fractions and sera as described in Section 2. Total = original serum. The arrows indicate in descending order, HDL, VLDL and LDL. In fractions 1-3 a faint band is seen above HDL: this is albumin.

slow acceleration and deceleration (program 6). Blank gradients were run simultaneously (50% iodixanol diluted with buffered saline) for refractive index and density determination. 2.2.2. Method 2

Serum (6 ml) was mixed with 50% iodixanol (2 ml) to give a final concentration of Iodixanol of 12.5% (w/v) and 5 ml was transferred to g-maxTM tubes (6.3 ml volume) for the VTi 65.1 vertical rotor. The samples were underlaid with a cushion of 20% (w/v) iodixanol (0.5 ml) before the tubes were filled by overlaying with buffered saline. The tubes were centrifuged at 33 000 g,, (65 000 rpm) at 16°C for 3 h in a Beckman L8-80 or L7 ultracentrifuge using slow acceleration to 600 rpm and no brake during deceleration from 1000 rpm.

2.2.3. Method 3

Larger volumes of serum were run in OptisealTM tubes (11.2 ml); 10 ml of serum in 12.5% iodixanol (w/v) was underlaid with a 0.5 ml of 30% (w/v) cushion and topped up with buffered saline. Centrifugation was as in Method 2.

2.2.4. Method 4 (two step gradient)

Five millilitres of serum in 12.5% iodixanol (w/v) was layered under 5 ml of 6’,; (w/v) iodixanol (1 vol of OptiprepTM plus 9 vol of buffered saline or serum) in an OptisealTM tube (11.2 ml) and topped up with buffered saline. Tubes were centrifuged as in Method 2

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total

7

1

2

3

4

5 6

8 9 10.11

fop

bottom

ADoliDoDrotein

total

1

12

Fraction

number

4

7

Al

2

3

5 6

8 9 10 11 12

bottom

top Fraction

number

Fig. 2. Apolipoprotein B and Al in fractions separated on iodixanol gradients. Serum was fractionated as described in the legend to Fig. 1. Each of the top 12 fractions was mixed with an equal volume of sample buffer and 10 ~1 of the first 9 fractions and 3 ,uI of the next three fractions was applied to wells of a 3-20% gel. Proteins were separated by electrophoresis, transferred to nitrocellulose membranes and immunoblotted using either anti-apo-B (upper panel) or anti-apo-Al (lower panel) as described in Section 2. Total = original serum.

2.3. Gradient collection

Quickseal and g-maxTM tubes (Methods 1 and 2) were unloaded dense end first by tube puncture (Beckman Fraction Recovery System) in 0.25 ml fractions except for the first two or three fractions (0.5 ml) which contained the plasma proteins at high concentration and were very viscous. OptisealTM tubes (Methods 3 and 4) were unloaded light end first in 0.5 ml fractions by upward displacement with Maxidens using a Nycomed Gradient Unloader. 2.4. Agarose electrophoresis

Lipoproteins in whole serum and gradient fractions (2 ~1) were analysed by electrophoresis within 24 h of preparation on preformed Hydrogel lipo + Lp(a) agarose gels on a LKB Sebia horizontal electrophoresis apparatus run at room temperature at constant voltage (50 v) for 90 min.

The running buffer which is supplied with the Hydrogel kits contained Tris 7.2 g/l, barbital 1.84 g/l, sodium barbital 10.30 g/l and sodium azide 0.1 g/l. The gels were dried, stained (15 min) with Sudan Black (160 ml absolute alcohol, 140 ml distilled water and 2 ml of the Sudan Black concentrated solution provided with the Hydrogel kits), and destained (5 min) in 45% alcohol. 2.5. Separation of plasma lipoproteins on salt gradients

Plasma lipoprotein classes were separated on discontinuous salt gradients or by sequential flotation essentially as described by Have1 et al. [14] for comparison with those separated on iodixanol. 2.6. Discontinuous salt gradients

The density of the serum was adjusted to 1.21 with solid KBr; 3 ml were pipetted into tubes for

J.M. Graham et al. /Atherosclerosis

the Beckman SW41 rotor and overlaid with 3 ml each of solutions of p = 1.15, 1.063 and 1.006 g/ml. These were prepared by mixing 0.9% NaCl (p = 1.006 g/ml) with a solution made up of 15.8% NaCl and 34.5% KBr (p = 1.34 g/ml). The tubes were centrifuged at 200000 g,, for 24 h and 6 x 2 ml fractions were removed from the top of each gradient. Fractions were dialysed against buffered saline for 16 h and aliquots were used to separate lipoproteins on Hydrogels, or for lipid analysis, or were diluted with buffered saline, mixed with 50% iodixanol to give a final concentration of 12.5% (w/v) and centrifuged according to Method 1. All solutions contained 1% disodium EDTA, 0.02% sodium azide and the salt gradients also contained 20 ,uM PMSF. Density (g/ml)

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129

2.7. Sequential centrifugation The density of the serum was adjusted to 1.O19 g/ml with solid KBr and centrifuged for 20 h at 200 000 g,, in the SW41 rotor. The top 1.5 ml (VLDL) was removed and the infranatant adjusted to density 1.063 g/ml and re-centrifuged; the top 1.5 ml (LDL) was again removed and the infranatant adjusted to 1.21 g/ml and re-centrifuged and the top 1.5 ml removed (HDL). All lipoprotein fractions were dialysed against buffered saline containing 1% disodium EDTA and 0.02% sodium azide for 16 h and aliquots were used to separate lipoproteins on Hydrogels or for lipid analysis. 2.8. SDS-PAGE and immunoblotting Aliquots of serum or gradient fractions were mixed with an equal volume of sample buffer and aliquots (2-10 ~1) were separated by SDS-PAGE on 3-20% gradients as described previously [13]. Proteins were transferred to nitrocellulose and immunoblotted for apo-B using MAC 131 and for apo-Al using anti human apo-Al as described previously [ 131.

1’.,

2.9. Analysis of lipid 1:

I

I

I

I

I

I

I

I

I

I

I

I

I1

1 2 3 4 5 6 7 8 9 1011 1213141516 Fraction Number Fig. 3. Iodixanol density profiles in the TLNIOO rotor. The starting concentration of iodixanol was 12.5% (w/v) and the tubes were prepared and centrifuged as described in Method 1 for 1 h (squares), 2 h (circles) and 3 h (triangles). Gradients were collected in 0.25 ml fractions from the bottom of the

The cholesterol and triacylglycerol content of serum and gradient fractions was determined using kits and standards supplied by Boehringer. The method was adapted to 96-well plates: 200 ,ul of reagent per well 2-10 ~1 aliquots of serum or gradient fractions were added and the colour development read at 450 nm using an Anthos HTll plate reader. A standard curve (l-5 ~1 of cholesterol standard containing 4.75 mmol/l or triacylglycerol standard containing 1.71 mmol/l) with and without 20% iodixanol was run on each plate. Inclusion of iodixanol had no effect on the standard curves. The mean absorbance for cholesterol per ~1 was 0.054 rf: 0.02 in the absence of iodixanol and 0.052 _+ 0.01 in the presence of iodixanol. The absorbance for triacylglycerol per ~1 was 0.128 _+ 0.008 in the absence of iodixanol and 0.130 + 0.01 in the presence of iodixanol.

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3456789.

bottom

Fraction Number

Fig. 4. Separation of plasma lipoproteins on iodixanol gradients using the VTi 65.1 rotor. Lipoproteins were separated on gradients of iodixanol using Method 2 as described in Section 2. Fractions were taken from the bottom of the gradient; the first three fractions, containing the concentrated plasma proteins, were 0.5 ml and subsequent fractions 0.25 ml. The three arrows indicate in descending order HDL, VLDL and LDL.

3. Results

3.1. Separation of lipoproteins using the TLNlOO rotor Analysis of gradient fractions (Method 1) by agarose electrophoresis showed that VLDL was concentrated in the top fraction (fraction 14) of the gradient, LDL was in fractions 7- 13 with a peak in 9-10 and HDL was in fractions 2-7 with a peak in fraction 5 (Fig. 1). There was a slight overlap between LDL and HDL in the middle of the gradient, however, the bulk of the two lipoprotein classes were well separated and pure LDL or HDL could be collected excluding the region of overlap in the middle of the gradient. Before harvesting it was possible to see in the gradients an opalescent top band corresponding to VLDL, an orange band (about one third of the way down the tube) corresponding to the peak of LDL, and a yellow-orange band corresponding to the peak of HDL immediately above the viscous plasma proteins, which occupied approximately 1 ml at the bottom of the tube. The cholesterol profile of the gradient showed a major peak corresponding to the LDL fraction and a minor peak coincident with HDL, and the triacylglycerol was largely concentrated in the VLDL fraction (Fig. 1). To test its general applicability we used the same method to separate plasma samples with cholesterol levels

ranging from 5.0-7.3 mmol/l and triacylglycerol levels from 1.8-4.3 mmol/l. Similar patterns in the lipoprotein separation and lipid distribution were observed, although there were differences in the amounts of lipoproteins in individual fractions. In six different plasma samples separated using Method 1 the recovery of cholesterol from the gradient was 99.66% + 3.16% (S.E.M.) and that of triacylglycerol was 96.82% f 7.56% (S.E.M.) and, compared with the original serum, the maximum enrichment of the cholesterol was 5.0 fold in the LDL peak, and of the triacylglycerol was 6.8 fold in the VLDL fraction. Using immunoblotting apo-B was found to be concentrated in the LDL-containing fractions and apo-Al in the HDL-containing fractions (Fig. 2). In the case of apo-B the apparent double band in the most concentrated fractions is an artefact of overloading which was not apparent on the original gel. For direct comparison of fractions similar aliquots were used, which resulted in overloading of the most concentrated samples. To separate the different lipoprotein classes a 3 h run time was selected; however, depending on the separation required, the density profile of the gradient can be modulated by changing the centrifugation parameters. With increasing time the profile changes predictably from an S-shape with a very shallow mid-region (1 h) to a more linear profile (2 h) and then to a more or less exponential profile (3 h) (Fig. 3).

131

1

3

5

7

9 Fraction

11

13

15

17

19

Number

Fig. 5. Separation of plasma lipoproteins on iodixanol gradients using OptisealTMtubes in the VTi 65.1 rotor. Lipoproteins were separated on gradients of iodixanol using Method 3 (A) or Method 4 (B) as described in Section 2. Fractions (0.5 ml) were taken from the top of the gradient. The three arrows on the electrophoresis panel indicate in descending order HDL, VLDL and LDL. Blank gradients were performed simultaneously and the density (g/ml) determined from the refractive index.

3.2. Separation cf lipoproteins using the VTi 65.1 rotor The basic separation method can be adapted to the VTi 65.1 vertical rotor. Method 2 used gmaxTMtubes which hold 5 ml of sample compared

with 3.5 ml in the TLNlOO rotor. A cushion of 20% iodixanol was included to prevent any soluble proteins sedimenting to the wall of the tube. Although the path-lengths of the TLNlOO and the VTi 65.1 rotors are similar, the gradient is spread out on re-orientation because of the greater tube

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1 2 top

3

total 1 2

4

3

5

4

6

5

7

6

6

124 (1996) 125-135

9

10

11

12

12

13 14 bottom

3

Fig. 6. Comparison of plasma lipoproteins separated on iodixanol and salt gradients. Plasma from the same individual was separated on an iodixanol gradient using Method 1, on discontinuous salt gradients, and by sequential centrifugation as described in Section 2. Aliquots of the fractions (2 or 3 ~1) were separated on Hydragel agarose gels. a. Fractions I- 14 from iodixanol gradients b. Fractions from discontinuous salt gradient: total = original plasma and fractions l-6 and c. Lipoproteins separated by sequential centrifugation 1 = VLDL, 2 = LDL and 3 = HDL.

height of the g-maxTMtubes and a greater number of fractions were taken. Despite these operational differences, similar overall separations were obtained using Method 2 as those with Method 1 (Fig. 4). The main difference was that the HDL were spread into a greater number of fractions. VLDL were in the top fraction and LDL in the next 7-8 fractions as in Method 1. In the 11.2 ml OptisealTMtubes using Method 3, VLDL again banded at the top of the gradient, although there was some tailing into the LDL zone (fractions 3-6). The HDL banded broadly in fractions 8-18 (Fig. 5A). Using a two step gradient (6/12.5% iodixanol - Method 4) the LDL were spread-out in the top half of the gradient (fractions 3-10) while the HDL zone was compressed in the denser part of the gradient (Fig. 5B). Identical results were obtained when the plasma was only in the bottom layer or in both layers of the gradient. The banding density of VLDL was < 1.006 g/ml; of the LDL, 1.01-1.030 g/ml; and of the HDL, 1.030-1.14 g/ml (Fig. 5). These are lower than the densities in salt gradients - LDL, 1.0191.063 g/ml and HDL, 1.0633 1.21 g/ml. Because

the iodixanol gradients are essentially iso-osmotic, protein molecules will maintain their native hydration, in contrast to their loss of water (and consequent increase in density) in highly hyper-osmotic salt gradients. The lipoproteins in different fractions separated in Methods l-4 (Figs. 1, 4 and 5) exhibited slightly different relative electrophoretic mobilities suggesting that subfractionation of LDL and HDL based on small differences in density may occur on the iodixanol gradient. This may also account for VLDL occurring in several fractions in Method 4 (Fig. 5). 3.3. Comparison of lipoproteins separated in salt gradients with those separated on iodixanol

Separation of lipoproteins on iodixanol was directly compared with separation using conventional salt gradients. Centrifugation of serum from the same individual on iodixanol or discontinuous salt gradients yielded good separations of lipoproteins based on electrophoretic mobility on Hydrogel agarose gels (Fig. 6a and Fig. 6b). On the salt gradients VLDL was concentrated in frac-

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Table 1 Recoveries of cholesterol and triacylglycerol in lipoprotein fractions separated on iodixanol, salt gradients or by sequential centrifugation on salt solutions Iodixanol

VLDL LDL HDL Plasma Recovery

Salt gradient

Sequential

CHOL pmoles

TAG ,~moles

CHOL pmoles

TAG pmoles

CHOL p umoles

TAG pmoles

0.59 * 10.62 & 4.88 & 16.26 2 98.01%

2.15 f 0.81 & 0.52 k 3.63 f 95.87%

0.64 & 5.95 + 2.00 + 16.26 i 65.12%

2.03 k 0.44 * 0.20 f 3.63 + 62.18%

0.96 i 3.38 i 1.63 + 16.26 & 36.72%

2.55 + 0.39 + 0.24 + 3.63 k 80.95%

0.07 0.20 0.39 0.51

0.06 0.02 0.15 0.45

0.10 1.06 0.43 0.51

0.12 0.05 0.05 0.45

0.13 0.60 0.16 0.51

0.15 0.20 0.095 0.45

Lipoproteins from the same serum samples were separated on iodixanol gradients, discontinuous salt gradient or by sequential centrifugation on salt solutions and the cholesterol and triacylglycerol content determined as described in Section 2. The distribution of the lipoproteins is shown in Fig. 6. The total recovery of triacylglycerol (TAG) and cholesterol (CHOL) in each lipoprotein class from 3 ml serum starting volume was calculated taking into account the final dilution of the fraction. For the fractions from the iodixanol gradient the total lipid in VLDL was that in fraction 1; the total lipid in LDL was the sum of fractions 2-8 and in the HDL the sum of fractions 9- 14. For the fractions from the discontinuous salt gradient the total lipid in VLDL was fraction 1, LDL fraction 2 and HDL the sum of fractions 446. Recoveries are expressed as %, of lipid in the original serum sample separated recovered in all fractions collected. Results are from separations of the same serum and are the mean of 4 determinations k S.D.

a

b

1

2

3

4

5

6

Fraction

7

6

9

number

10 11 12 13 14 bottom

Fig. 7. Separation on iodixanol gradients of plasma lipoproteins separated on discontinuous gradients. Fractions 1 (VLDL), 2 (LDL) and 4 + 5 (HDL) from the discontinous salt gradient illustrated in Fig. 6b were diluted to 3 ml with buffered saline and mixed with 1.Oml of 50% iodixanol and centrifuged as for Method 1. Fractions were collected from the gradient and separated on agarose gels as described in Section 2. a = VLDL, b = LDL and c = HDL.

tion 1, LDL in fraction 2 and HDL in fractions 4-5 with an overlap of LDL and HDL in fraction 3. Separation of serum using sequential centrifugation separated VLDL and LDL effectively but

the HDL fraction contained a large amount of LDL (Fig. 6~). When the fractions corresponding to VLDL, LDL and HDL from the discontinuous salt gradient were re-centrifuged on iodixanol gra-

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dients as for Method 1 the fractions moved to essentially the same positions on the gradient as those in total serum (Fig. 7). Most of the VLDL was in fraction 1, although there was some tailing into fractions 2 and 3. This may be due to some degradation during the long time period (about 2 days) required to prepare the lipoprotein fractions. LDL was in the fractions 2-8, while HDL was in fractions 7-12. Because of the differences in the method of separation and processing of the lipoprotein fractions from iodixanol gradients, salt gradients and sequential centrifugation it is not possible to compare the concentrations of the lipoprotein containing fractions directly. However, taking into account the volume of serum separated and the final dilutions of each of the fractions prepared the amounts of cholesterol and triacylglycerol recovered in each lipoprotein class can be calculated (Table 1). The relative triacylglycerol and cholesterol compositions of VLDL, LDL and HDL separated by all three methods were similar, however, the recoveries of lipids in fractions from the iodixanol gradient were considerable greater that those using the other two methods.

4. Discussion

In the methods described we have taken advantage of the ability of iodixanol to form self-generating gradients whose density profiles can be made shallow in the p = l.OlO- 1.100 g/ml region and the availability of short sedimentation path length vertical and near-vertical rotors which increase the efficiency of gradient formation. The major classes of plasma lipoproteins are effectively resolved and there are indications from the electrophoretic mobility data that subfractions of lipoproteins can be resolved; this is under further investigation. Lipoproteins prepared in iodixanol gradients are similar to those prepared by conventional salt gradients and fractions from the latter behave as expected when centrifuged in iodixanol. However, there are significant advantages to using iodixanol:

124 (1996) 125-135

(1) as the gradients are self-generating, filling the centrifuge tubes is technically easier and faster than layering salt gradients; (2) the gradients are highly reproducible, stable and easily modulated; (3) the centrifugation time is 3 h compared with 20 h for the single step salt gradients or 4 days for the sequential method; (4) as iodixanol is inert and non-ionic, fractions can be analysed directly so minimising losses due to handling; (5) the concentration of lipoproteins achieved by the gradients increases the sensitivity of the analysis. Depending on the aim of the investigation and the equipment available the basic method described here can be modified as required. We selected a 3 h centrifugation step in the current study because this provided a relatively shallow gradient profile over a wide density range suitable for fractionation of VLDL, LDL and HDL. However, the gradient profile of iodixanol, and hence the separation profile of the lipoproteins, can be varied by changing the speed or time of centrifugation and/or the initial concentration of iodixanol. Thus, the VLDL, LDL or HDL fractions can be spread on the gradient to enhance the separation of different subclassesby changing the centrifugation time (see Fig. 3) or by changing the gradient itself (see Figs. 4 and 5a and Fig. 5b). If removal of plasma proteins is important then a two step gradient in which the lipoproteins are floated from a load layer as in Method 4 would be preferable. The basic method described here can be used preparatively for separation of relatively large amounts of plasma (up to 60 ml using the VTi 65.1 rotor) If pure fractions of VLDL, LDL or HDL are required the appropriate fractions can be pooled excluding the small region of overlap, or the gradient can be modified to produce complete separation of the lipoprotein of interest. In addition, the new method can be used analytically for obtaining lipoprotein/apolipoprotein/cholesterol/triacylglycerol profiles and has potential as a new method for obtaining plasma lipoprotein profiles of individuals after initial screening meth-

J.M. Graham et al. i Atherosclerosis

ods have indicated some abnormality. The rapid separation of lipoproteins in the TLNlOO will also be advantageous in analysis of the lipid and vitamin composition of lipoprotein classeswhich are susceptible to oxidative damage in prolonged centrifugation and dialysis.

Acknowledgements We would like to thank the BBSRC, and the Medical Research Council for research support.

References [l] Mackness M, Durrington PN. Lipoprotein separation and analysis for clinical studies. In: Converse CA and Skinner ER. (eds). Lipoprotein Analysis: A Practical Approach. IRL Press at Oxford University, Oxford, UK 1992;l. [2] Lindren FT, Jensen LC, Hatch FT. Isolation and quantitative analysis of serum lipoproteins. In: Nelson GJ, (ed), Blood Lipids and Lipoproteins, Quantitation, Composition and Metabolism. R.E. Krieger Publishing Co. Huntington, NY 1979;181. [3] Chapman M, Goldstein J, Lagrange D, Laplaud PM. A density gradient ultracentrifugation procedure for the isolation of the major lipoprotein classesfrom human serum. J Lipid Res 1981;22:339. [4] Kelley JL. Kruski AW. Density gradient ultracentrifugation of serum lipoproteins in a swinging bucket rotor. Methods Enzymol 1986;128:170.

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of density gradient centrifugation for the separation of serum lipoproteins. Clin Chim Acta 1974;53:355. PI Chung BH, Segrest JP, Ray MJ, Brunzell JD, Hokanson JE, Krauss RM, Beaudrie K, Cone JT. Single vertical spin density gradient ultracentrifugation. Methods Enzymol 1986;128:181. [71 Kulkarni KR, Garber DW. Marcovina SM, Segrest J.P. Quantitation of cholesterol in all lipoprotein classes by the VAP-11 method. J Lipid Res 1994;35:159. PI Timasheff SN. The control of protein stability and associations by weak interactions with water. Annu Rev Biophys Biomol Struct 1993;22:67. [91 Ford T, Graham JM, Rickwood D. Iodixanol: a nonionic iso-osmotic centrifugation medium for the formation of self-generating gradients. Anal Biochem 1994;220:360. [lOI Jorgenson NP, Nossen JO, Borch KW, Kristiansen AB, Kristoffersen DT, Lundby B, Theodorsen L. Safety and tolerability of iodixanol in healthy volunteers with reference to two monomeric X-ray contrast media. Em J Radio1 1992;15:252. Ull Bostad B, Borch KW, Grynne BH, Lundby B, Nossen JO, Kloster YF, Kristolfersen DT, Andrew E. Clincal trials of newer iodinated agents, safety and tolerability of iodixanol. lnvest Radio1 1991;26:S201. WI Gherardi E, Hutchings A, Galfre G, Bowyer DE. Rat monoclonal antibodies to rabbit and human low density lipoproteins. Biochem J 1988;252:237. v31 Wilkinson J. Higgins JA, Groot PHE, Gherardi E, Bowyer DE. Determination of the intracellular distribution and pool sizes of apolipoprotein B in rabbit liver. Biochem J 288;1992:413. [I41 Have1 RJ. Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 1955;33:1345.