Separation of human plasma lipoproteins by density gradient

centrifugation in iodixanol

Professor David BillingtonSchool of Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UKEmail: d.billington@ljmu.ac.uk

Composition and density of human plasma lipoproteins

252045101.006-1.063LDL

5-1015-2010-4040-800.95-1.006VLDL

Approx composition(% dry weight)

Density (g/ml)Name

45-5030201-51.063-1.21HDL

1-23-62-580-95<0.95Chylomicron

Prot.PL.Chol.TAG.

Fractionation of human plasma lipoproteins by sequential flotation

Chylomicron-free plasma (1.03 g/ml)

100,000g

24 h

saline

>>1.03 g/ml

1.006 g/mlVLDL

KBr

1.063 g/mlLDL

1.21 g/ml

KBr

HDL

Iodixanol - a medium for self-generated density gradients

CONHCH2 CHCH2 OH

OH

CONHCH2 CHCH2 OH

OH

NCH2 CHCH2 N

OHCO CO

CH3 CH3

CHCH2 NHCO CONHCH2 CH

CH2 OH CH2 OH

OH OH

I

I

II

I

I

MWt 1550

Properties of iodixanol

• Non-ionic• Non-toxic

• Clinically tested as an X-ray imaging agent

• Commercially available as a 60% solution - OptiPrep™

Self-generated gradients • At g-forces above 180,000g iodixanol

molecules will start to sedimentg

time

Axis ofrotation

Vertical

Swinging-bucket

Sedimentation path length of rotors

Self-generated gradient strategy

1g 1g

350,000g350,000g

Common Beckman near-vertical and vertical rotors

400,0008 x 2.0 mlTLV100

450,0008 x 3.3 mlTLN100

645,000416,000402,000242,000

8 x 4.9ml16 x 4.9 ml8 x 11.2 ml8 x 36.2 ml

VTi90/NVT90VTi65.2/NVT65.2VTi65.1/NVT65VTi50

Maximum gCapacityRotor

170,000g353,000g

Fraction Number

Density (g/ml)

1 3 5 7 9 111

1.1

1.2

VTi65.1 rotor; 3h

Start: 12% iodixanol (1.08 g/ml)

Lipoprotein fractionation strategy

Blood Plasma

2000g

20 min100,000g

10 min

Chylomicrons

LDLVLDL

HDL350,000g

2.5-3.0h

Saline

12%iodixanol

6% or 9%iodixanol

P

2

P

3

P

4

P

1

C

M

EP

Unloading densitygradients with a Labconco AutoDensi Flow

15 13 11 9 7 5 3 1

8

6

4

2

0

1.6

1.2

0.8

0.4

0

Cho

lest

erol

(mM

)

TA

G (m

M)

Fraction numberbottom top

Typical fractionation in 12% iodixanol in a TLN100 rotor

From: Higgins JA, Graham, JM & Davies I (2001) In Atherosclerosis, ExperimentalMethods and Protocols (ed Drew AF) Humana Press, Totowa, NJ pp 37-49.

HDL LDL VLDL

Banding Densities in Iodixanol

1.03-1.141.063-1.210HDL

1.01-1.031.019-1.063LDL

<1.006<1.006VLDL

Density in iodixanol (g/ml)

Density in KBr(g/ml)

Lipoprotein

LDLs consist of lipid and protein and range from a small dense, protein-rich particle to a more buoyant, lipid-rich one.

Small, dense LDL

Large, buoyant LDL

BB

B

B

Small dense, atherogenic LDL

Atherogenic, associated with 3 x > risk of CVD

0.20

0.10

1.30

1.20

1.10

1.000

44 Fractions

mol

es/fr

actio

n

Den

sity

(g/m

l)

Cholesterol profiles of three subjects in 9/12% iodixanolself-generated gradients in TLN100 rotor

From Sawle, A, Higgins, MK, Olivant, MP,and Higgins, JA (2002)J. Lipid Res. 43, 335-343.

topbottom

340,000g/3h

1D Gel Scanner

Fractionation and analysis of Coomassie blue stainedlipoproteins in NVT65 rotor

From Davies, IG and Griffin BA (2001) British Hyperlipidaemia AssociationEdinburgh, July 2001; Atherosclerosis (2001) 159, 247-252.

9%

12%3 ml

8 ml

12 3

DensityReferencescale

12

3

0.280.33 0.42

0.53

Analysis of Coomassie-blue stained LDL bands

From Davies, IG and Griffin BA (2001) British Hyperlipidaemia AssociationEdinburgh, July 2001; Atherosclerosis (2001) 159, 247-252.

VTi50 rotor (8 x 39 ml): 5h: 206,000g

12%

6%

6/12%

9%

Large Scale Preparation of lipoproteins

Separation of plasmalipoproteins in a VTi50 rotor

TAG

Chol

apoB

apoA

1

2

3

4

5HO

6HO

OH

7HO

OH

β-carotene

α-carotene

all-trans lycopene

5-cis lycopene

-cryptoxanthin

zeaxanthin

lutein

Carotenes

X Xanthophylls

min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

Norm.

-5

0

5

10

15

DAD1 A, Sig=460,4 Ref=off (DAN\PLASMG24.D)

3.5

43 3

.693

4.6

41 4

.880

5.0

70

5.4

92 5

.666

5.9

74

6.5

91

7.1

85 7

.447 8

.727

9.0

50

11.

532

17.

102

17.

882

Separation of plasma carotenoids on C18 column:

Source of extract : 1ml of plasma from female, aged 41, non-smoker

Extraction : 1ml ethanol, vortex, 1.5ml ethylt ether, vortex, 1.5ml hexane, vortex, take off top layer

Sample : dried under OFN at Rt and resuspended in 200µl THF/Methanol 10/90

System : Agilent 1100 Series LC Chemstation

Column : Phenomenex Gemini 5µm C18 110A 4.6 x 250mm

Mobile Phase : 66/22/10 Acetonitrile/THF/Methanol (with ammonium acetate)

Temp : 22oC

Flow Rate: 0.8 ml/min

Injection Volume : 30µl

lutein and zeaxanthin lycopene α- and β-carotene

0

5

10

15

0 2 4 6 8 10 12 14 16Fraction Number

% b

-Cry

ptox

anth

in

0

5

10

15

20

0 2 4 6 8 10 12 14 16Fraction Number

% α

-Car

oten

e

0

5

10

15

20

0 2 4 6 8 10 12 14 16Fraction Number

% β

-car

oten

e

0

5

10

15

20

25

0 2 4 6 8 10 12 14 16

Fraction Number

% L

ycop

ene

0

5

10

15

20

25

0 2 4 6 8 10 12 14 16Fraction Number

% 5

-cis

Lyc

open

e

0

5

10

15

0 2 4 6 8 10 12 14 16Fraction Number

% L

utei

n +

Zeax

anth

in

β-carotene α-carotene

all-translycopene

5-cislycopene

β-cryptoxanthin lutein +zeaxanthin

Carotenoid distribution in lipoprotein fractions

0

10

20

30

40

50

60

70

80

90

0 2 4 6 8 10 12 14 16

Fraction Number

% c

arot

enoi

d in

Eac

h Fr

actio

n

LDLVLDL HDL

carotenes

xanthophylls

Carotenes are preferentially located in LDL and VLDLXanthophylls are preferentially located in HDL

0

5

10

15

20

25

0 2 4 6 8 10 12 14 16 18

Fraction Number

% C

hole

ster

ol in

Eac

h Fr

actio

n

0

5

10

15

20

25

0 2 4 6 8 10 12 14 16 18

Fraction Number

% β

-Car

oten

e in

Eac

h Fr

actio

n

0

5

10

15

20

25

0 2 4 6 8 10 12 14 16 18

Fraction Number

% a

ll-tra

ns L

ycop

ene

in E

ach

Frac

tion

0

5

10

15

20

25

30

0 2 4 6 8 10 12 14 16 18

Fraction Number

% 5

-cis

Lyc

open

e in

Eac

h Fr

actio

n

Cholesterol β-carotene

all-trans lycopene 5-cis lycopene

Small dense LDL particles are not depleted of carotenes

Advantages of iodixanol

• Lipoproteins close to their native state• Self-generated gradients simple and

reproducible• Close to 100% recovery of lipids• Identification and quantitation of the

principal LDL subclasses and their components

• Potential for adaptation of techniques to HDL and VLDL subfractionation

With thanks to:

• Gordon Lowe • Ian Davies• John Graham and Terry Ford• Andy Young and Danny Graham

(in no particular order)