Separation of human plasma lipoproteins by density ... · lipoproteins by density gradient...
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Transcript of Separation of human plasma lipoproteins by density ... · lipoproteins by density gradient...
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: [email protected]
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)