Improved Serum Lipoprotein Profiling by cITP Using a

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User ID: 11913
Improved serum lipoprotein profiling by cITP using a novel spacers mixture
Background
Lipoproteins are the main carriers of lipids in blood. Determining the lipoprotein profile in
blood is vital to gain insight into cardiovascular disease (CVD), considering that low density
lipoproteins (LDL), particularly their oxydated or glycated forms, are regarded as atherogenic and that
high density lipoproteins (HDL) may play an anti-atherogenic role. Analytical methods with improved
separation capabilities are required for more detailed and informative lipoprotein profiling in blood
serum. Capillary isotachophoresis (cITP) renders the technique with the most promising analytical
capabilities in this case [1, 2]. An appropriate selection of spacers is crucial on the separation outcome
and previous papers pay little attention to this fact. The goal of this work was to develop an improved
separation method for lipoproteins based on cITP.
Methods
cITP separations were performed on blood serum pre-stained with Sudan Black B, mixed with
spacers and leading electrolyte (LE)- 10 mM HCl, 0.35% hydroxypropylmethylcellulose (HPMC),
adjusted to pH 8.8 or 9.0 with ammediol. 39 spacer compounds were selected upon their theoretical
electrophoretic mobilities and tested. Terminating electrolyte (TE) contained 20 mM L-Ala, pH 9.4 or
9.0 adjusted with Ba(OH)2/ammediol and alternatively 0.35% HPMC. Fresh blood serum obtained
from a healthy volunteer was used for isolation of lipoprotein reference fractions (HDL, LDL, VLDL)
by density gradient ultracentrifugation. Analysis of serum or lipoprotein-deficient serum spiked with
the lipoprotein reference fractions was carried out for assignment of the separated lipoprotein species.
A capillary electrophoresis instrument equipped with a UV-vis detector was employed and doublycoated methylated fused silica capillaries were used.
Results
A mixture of 24 spacers was selected out of the tested spacers (mainly aminoacids, dipeptides
and sulphonic acids), for a better ITP separation of lipoproteins at pH 8.8/9.4, giving rise to 17
separated species (Figure 1), surpassing previously reported cITP methods. Lipoprotein-spiked serum
analysis showed that HDL is found within the first 10 peaks, VLDL is mainly located in peaks 11-12,
while LDL is distributed within peaks 12-17. LDL and HDL subpopulation separation was therefore
considerably improved. A pH gradient in the LE/TE seems to be more appropriate for the lipoproteins
separations (a higher degree of comigration of VLDL and LDL is observed when the separation is
performed instead at constant pH 9, Figure 2).
The doubly-coated methylated capillaries used for the separation were functional for at least
100 runs and absolute migration times were typically lower than 3% for 25 repeated runs (Table 1).
This improves considerably the performance described on previous works, where long-term capillary
stability at high pH is an issue.
Inclusion of HPMC in the TE has no effect in the separation and makes feasible, when
required, the controlled pressure-driven fraction collection of separated lipoproteins with conventional
CE systems.
Conclusions
We have developed an improved lipoproteins separation method at analytical scale with a
promising potential for a more detailed study of subparticle populations and fractionation possibilities
in CVD patients serum.
This work is unpublished.
1.
2.
A. Böttcher, J Lipid Res, 2000. 41(6): p. 905-915.
G. Schmitz, Electrophoresis, 1997. 18(10): p. 1807-1813.
Word count (technical description): 499 words
Oral presentation – CE applications
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DAD1 E, Sig=570,10 Ref=off (ESTEFANIA\200514-OV3-CH3-ITP-SER-MIX40-30.D)
12
Norm.
300
VLDL
LDL
VLDL
250
11
LDL
200
15
150
HDL
100
2
50
1
LDL
4 5
3
6
LDL17
LDL LDL
78
13 14 16
9 10
0
6
6.5
7
7.5
8
8.5
9
9.5
10
min
Figure 1. cITP electropherogram showing the lipoprotein separation profile using a leading electrolyte
at pH 8.8 and terminating electrolyte at pH 9.4. Peak assignment: HDL is found in peaks 1-10. VLDL
is found in 11-12 and a slight amount might also be present in peak 10. LDL appears in peaks 12-17.
Detection at =570 nm.
DAD1 E, Sig=570,10 Ref=off (ESTEFANIA\200514-OV3-CH3-ITP-SER-MIX40-9-37.D)
mAU
VLDL
14
11
LDL
VLDL
250
LDL
VLDL
200
12
LDL
16
150
LDL
HDL
13
100
50
1
2 3
4
7
5
6
LDL
15
8 10
9
LDL
17
0
5
5.5
6
6.5
7
7.5
8
8.5
min
Figure 2. cITP electropherogram showing the lipoprotein separation profile using both leading
electrolyte and terminating electrolyte at pH 9.0. Peak assignment: HDL is found in peaks 1-10.
VLDL is found in 11,12,14. LDL is present in peaks 12-17. Detection at =570 nm.
Table 1 – Repeatibility of cITP lipoprotein separations at pH 8.8/9.4 using a doubly coated methylated
capillary. 25 consecutive injections were performed. Data on migration times for peaks 1, 11
and 17 are reported.
peak 1
peak 11
peak 17
Mean migration time, 25
measurements
6.88
8.47
9.38
Standard deviation, min
0.21
0.23
0.25
Coefficient of variation, %
3.00
2.67
2.66
Word count (technical description): 499 words