Vol.2, No.7, 722-730 (2010)
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Influence of poloxamer 407 on fractional and
subfractional composition of serum lipoproteins of mice
Tatyana A. Korolenko1*, Fedor V. Tuzikov2,3, Thomas P. Johnston4, Natalia A. Tuzikova2,3,
Elena E. Filjushina1, Viktoriya M. Loginova1, Natalia G. Savchenko1
1Institute of Physiology, Siberian Branch of Russian Academy of Medical Sciences, Novosibirsk, Russia;
*Corresponding Author: t.a.korolenko@physiol.ru
2Boreskov Institute of Catalysis, Siberian Branch of Russian Academy of Sciences, Novosibirsk, Russia
3Novosibirsk State University, Novosibirsk, Russia
4University of Missouri-Kansas City, Kansas City, USA
Received 4 March 2010; revised 22 March 2010; accepted 25 March 2010.
Using a novel small-angle X-ray scattering (SAXS)
method for determination of fractional and sub-
fractional composition of lipoproteins (LPs), a
significant elevation of total cholesterol-lipop-
roteins (C-LP) and, especially, total triglyceride-
lipoproteins (TG-LP), was shown in this work.
Among the LP fractions, poloxamer 407 was
shown to significantly increase proatherogenic
total C-LDL, TG-LDL and, especially, their pre-
cursors C-VLDL and TG-VLDL, while only ex-
hibiting a moderate increase in the antiathero-
genic C-HDL and TG-HDL fractions. With regard
to the VLDL subfractions, significant elevations
were observed in both subfractions studied;
namely, C-V LDL 1-2 and C-V LDL 3-5. Similar chang-
es were no ted in the TG-V LDL1-2 and TG- VLDL 3-5
subfractions. The C-IDL and TG-IDL subfrac-
tions were increased significantly (20- to 30-
fold), while the C-LDL1-3 subfraction was mod-
erately (3- to 5-fold) increased at 48 hrs and at
day 4. In the moderately elevated (2- to 4-fold)
anti-atherogenic HDL fraction, the C-HDL2 sub-
fraction was increased more significantly (4-
fold) compared to the C-HDL3 subfraction; how-
ever, both C-HDL subfractions returned to base-
line by day 4. The elevation in the TG-HDL2
subfraction was observed only at 24 hrs. Mouse
models of hyperlipidemia and atherosclerosis
are useful to evaluate the role of “individual”
LPs, as well as their fractions and subfractions,
in hyperlipidemia and the genesis of athero-
Keywords: Poloxamer P-407; Dyslipidemia;
Serum Lipoprotein Fractions and Subf r actions
Changes of different classes of circulating lipoproteins
are the important indicies of lipid metabolism in physi-
ology and pathology; lipoproteins have been shown to
also play a regulatory role in vivo [1,2]. The main lipo-
protein classes consist of pro-atherogenic low-density li-
poproteins (LDL), very-low-density lipoproteins (VLDL),
and anti-atherogenic high density lipoproteins (HDL),
and are widely used as common lipid biomarkers in
atherosclerosis [1]. However, the biological role of sub-
classes of lipoproteins is still under investigation. The
regulatory role of lipoproteins has been shown to relate
to many intracellular processes, primarily to plasma
membrane permeability and fluidity as the result of
changes in the concentration of plasma membrane cho-
lesterol, with subsequent modifications of receptors and
transmembrane proteins (transmitters). In this process,
the role of total HDL and HDL subfractions have espe-
cially high importance in connection with their unique
capacity to accept cholesterol from the plasma mem-
brane and transport them to other classes of lipoproteins.
Hyperlipidemia is one of the main risk factors in the
development of cardiovascular and cerebrovascular dis-
eases common in contemporary society. In addition to
the elevation of plasma LDL-cholesterol and VLDL-
cholesterol, hypertriglyceridemia is suggested to be an
additional independent risk factor for atherosclerosis and
coronary heart disease [3]. According to recent data,
atherosclerosis is not only a metabolic lipid disease, but
has also been considered to result from inflammation
(chronic inflammatory disease). Statins have been re-
ported to not only lower LDL by inhibiting the activity
T. A. Korolenko et al. / HEALTH 2 (2010) 722-730
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)
reductase, but also by reducing inflammation to endo-
thelial cells.
For the prevention of atherosclerosis, it is important to
study the mechanisms of the early effects of hyperlipi-
demia on different cell types involved in the pathogene-
sis of atherosclerosis. Therefore, it is necessary to know
the types of lipoproteins involved in hyperlipidemia at
the early stages of atherosclerosis. It is well known that
during the process of atherosclerosis, foam cells initially
trap the oxidized LDL molecules via scavenger receptors.
The oxidized LDL molecule is digested and transformed,
and is then presented to T lymphocytes, initiating the
classic immunological reaction, and subsequently stimu-
lates the inflammatory response. Formation of lipid-laden
cells (mainly smooth muscle cells and macrophages) is
followed by increased secretion of pro-inflammatory
cytokines (like IL-6) and other factors (several types of
matrix metalloproteases), which promote inflammation.
Statin treatment has a pleiotropic protective effect, not
only by inhibiting HMG-CoA reductase, but also by ex-
erting its anti-inflammatory action.
The changes of serum lipoprotein levels responsible
for pro- and anti-atherogenic action have been studied
earlier [2]. However, recently, with help of new methods
for characterizing different lipoprotein fractions and
subfractions, some new data have been obtained on their
role in the pathogenesis of atherosclerosis [4]. For this
reason, the investigation of experimental models of hy-
perlipidemia is useful.
Poloxamer 407 is a block copolymer comprised of
polyoxyethylene and polyoxypropylene units, which is
known for its biocompatibility and potential to deliver
different medications for a variety of disease states [5,6].
Following acute administration, poloxamer 407 was
shown to induce significant hyperlipidemia, a model
which has been used for testing several hypolipidemic
drugs (statins, fibrates, and nicotinic acid) [7-11]. With
chronic administration of poloxamer 407 (4 months) to
mice, fibrofatty aortic lesions are developed, which are
similar in size and number to those observed in classic
diet-induced mouse models of atherogenesis [12,13].
Hyperlipidemia in acute poloxamer 407-induced ad-
ministration to rodents was characterized by a dramatic
elevation of both plasma cholesterol and triglycerides,
which is usually not observed in patients with athero-
sclerosis. In the poloxamer 407 mouse model of athero-
sclerosis, the mechanism of cholesterol and TG elevation
is associated with inhibition of cholesterol 7-hydro-
xylase and lipoprotein lipase, respectively [13], and not
dependent on PPARα [14]. It was also shown that a sin-
gle injection of poloxamer 407 administration to mice
caused hypercholesterolemia by inducing transient cho-
lesterolgenesis and down-regulating low-density lipo-
protein receptor expression [6]. The mechanism of ele-
vation of different classes and, especially subfractions of
lipoproteins with pro- and anti-atherogenic effects, is
still not known.
Small-angle X-ray scattering (SAXS) is a small-angle
scattering technique where the elastic scattering of
X-rays (wavelength 0.1 to 0.2 nm) by a sample, which
has inhomogeneities in the nanometer range, is recorded
at very low angles. This angular range contains informa-
tion about the shape and size of macromolecules, char-
acteristic distances of partially ordered materials, pore
sizes, and other data. SAXS is capable of delivering
structural information of macromolecules between 5 and
25 nm, of repeat distances in partially ordered systems of
up to 150 nm. SAXS is used in the characterization of
various materials. In the case of biologic macromole-
cules, such as proteins, the advantage of SAXS, over
crystallography, is that a crystalline sample is not needed.
The materials can be solid or liquid and they can contain
solid, liquid, or gaseous domains (so-called particles) of
the same or another material in any combination. SAXS
is accurate, non-destructive, and usually requires only a
minimum of sample preparation.
The aim of the present investigation was to evaluate
the lipoprotein-cholesterol and lipoprotein-triglyceride
fractions and subfractions in hyperlipidemia induced by
a single dose of poloxamer 407 to mice. Our overarching
goal was to quantify the changes in the serum lipopro-
tein levels after poloxamer 407 treatment by utilizing a
novel technique; specifically, small-angle X-ray scatter-
ing (SAXS).
2.1. Materials
Male CBA/C57BL mice (breeding station of the Institute
of Cytology and Genetics, Russian Academy of Sciences,
Novosibirsk, Russia) having a body mass of 20-25 g
were used. Poloxamer P-407 (Pluronic F-127, Sigma)
was administered to mice, as a single, i.p. injection, in a
dose of 1000 mg/kg. The animals were decapitated at 3,
24, 48 hrs, and then at 4, 5, 7, 12, 14 days after a single
dose of poloxamer 407. All experiments followed the
official “Rules for the work involving experimental
animals” and “Ethical Committee Recommendations of
working with laboratory animals”.
Serum was obtained after centrifugation of blood sam-
ples at 3000 × g for 15 min at 4C (Eppendorf Centri-
fuge 5415R, Germany) and stored at –70C until analy-
sis of total cholesterol (C), triglycerides (TG) of lipo-
proteins (LP): C-LP, TG-LP, their fractions, and subfrac-
T. A. Korolenko et al. / HEALTH 2 (2010) 722-730
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
2.2. Small-Angle X-Ray Scattering (SAXS)
According to Otvos et al. (2002) [15], LP fractions were
divided into the following 4 main classes: high density
LP (HDL), low-density LP (LDL), very-low-density LP
(VLDL), and chylomicrons (the last ones were not de-
termined by the method used) or 7 subfractions: HDL3,
HDL2, LDL, Intermediate-Density LP–IDL, VLDL3-5,
VLDL1-2, chylomicrons. Interval borders of fractions and
subfractions (according to scale of sizes, ro) were the
same as was indicated by Otvos (2002) [15].
A novel method for determination of fractional com-
position of LPs using the small-angle X-ray scattering
approach (SAXS) was used [4]. This method is inexpen-
sive, quick, and capable of determining the relative con-
tent of different LP fractions, both as a size distribution
of various LP particles and as absolute units of the total
concentration of lipid in LP fractions. At the same time,
clinical diagnostic laboratories usually determine the
concentrations of individual lipids in LP fractions,
mainly TG and free cholesterol (FC), and often the total
concentration of TG, FC and high density lipoproteins
(HDL) free cholesterol.
Measurement of fractional and subfractional composi-
tion of serum LP fractions and subfractions.was pro-
vided according to a method described earlier [4]. SAXS
roentgenograms were obtained using a Siemens diffrac-
tometer (Germany) by the method of step-by-step scan-
ning with use of a goniometer and X-ray scintillation
detector. Small-angle roentgenograms were measured in
the angular range: h = 0.013 ÷ 0.22 A-1, where h = 4π ·
sin(θ)/λ; 2θ-scattering angle. A special thermostatted
(20oC) quartz capillary cuvette (0.6 mm in diameter),
having a wall thickness of 0.01 mm was used. The radia-
tion wavelength (λ) was 1.54 Е. The small-angle X-ray
roentgenograms were corrected by taking into account
background scattering, adsorption, and collimation, after
which the X-ray data became smoothed. The first step of
mathematical processing of the SAXS data and compu-
tation checks of functions for size distribution of spheri-
cal particles were executed using a special computer
program and algorithms described earlier, and also by
use of optimization programs [4].
The results are reported as the mean standard devia-
tion of at least 3 different experiments for each sample
analyzed. The differences between samples were ana-
lyzed by the Student’s t-test, and a P 0.05 was consid-
ered statistically significant.
2.3. Morphological Study of Liver
For morphometrical study of liver tissue, samples were
first fixed in a mixture of 2% paraformaldehyde and
2.5% glutaraldehyde on 0.1 M phosphate buffer, post-
fixed in 1% osmium tetroxide solution, and then em-
bedded in an Epon-Araldit mixture. Semithin, 1 micron
tissue sections were obtained using the Ultramicrotome
LKB-8800-V (Bromma, Vallingby, Sweden), stained with
toludine blue and examined under a STAR Zeiss light
microscope (Germany). The numeric density of macro-
phage cells was calculated as the number of cells per 1
мм2 of tissue at a final magnification of 640. Not less
than 50 fields of view were studied for every series of
experiments. The cell measurements were made with the
Motic Images 2000 Program. Ultrastructural changes of
liver cells were investigated with an electron microscope,
JEM 1400 (Jeol, Japan). Quantitative data were then
processed using the Statistica-4 program, and the reli-
ability of distinctions judged by the Student’s t-test.
The relative weight of liver and spleen increased 48 hrs
after poloxamer 407 administration (when the most sig-
nificant hyperlipidemia was noted) (Figure 1). In pe-
ripheral blood, there was an increased number of mono-
cytes observed at day 12 (Figure 2).
3.1. Morphological Studies
Light and electron microscopic studies demonstrated that
liver sinusoids were considerably extended 1 day after
poloxamer 407 administration, and that the numeric
density of macrophages was significantly increased (al-
most twice) (Table 1). The heterogeneity of macrophage
dimensions was strongly increased. Very large cells, with
X-axis: time after the single administration of poloxamer 407 (1000
mg/kg) to mice
Y-axis: Organ: Body weight ratio of liver and spleen, %.
The relative weight was expressed as the organ: body weight ratio
Control group n = 26, poloxamer-treated groups the mean± SEM rep-
resent a minimum of 7 mice at each time point.
The data are shown as mean ± SEM (*p < 0.01 vs control)
Figure 1. Relative liver and spleen weight of mice during sin-
gle administration of poloxamer 407 (1000 mg/kg) to mice.
T. A. Korolenko et al. / HEALTH 2 (2010) 722-730
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
X-axis: Time after poloxamer 407 administration to mice.
Y-axis: The number of PMN, monocytes and lymphocytes is expressed
as % from the total amount of leukocytes.
Poloxamer was administered i.p. as a single dose (1000 mg/kg).
The number of animals in each group is 5.
The data are shown as mean ± SEM (*p < 0.05 vs control).
Figure 2. Influence of poloxamer 407 on leukocytes composi-
tion of the peripheral blood of mice (%).
Table 1. The numerical density of liver macrophages during
single administration of poloxamer 407 to mice.
Groups, the number of mice The numeric density of liver
macrophages, per mm2
1. Control (intact) (5) 1277.4 ± 36.86
2. Poloxamer, 24 hrs (5) 2002.6 ± 36.58
P < 0.01
3. Poloxamer, 5 days after (5) 844.6 ± 28.74
P < 0.01
The data are shown as mean ± SEM.
The number of mice is in the parentheses.
a cross-sectional area more than 90-100 square microns,
were observed among macrophages, which comprised
about 40% of the macrophage population. These mac-
rophages were filled with granular material, so their cy-
toplasm had a foamy appearance (Figure 3).
Five days after poloxamer 407 administration, mor-
phometric data for the liver demonstrated that the spe-
cific numerical density of macrophages was decreased,
not only in comparison with the previous value, but in
relation to the controls as well (Table 1). As before,
large macrophages with a “foamy” cytoplasm containing
electron light (transparent) material (possibly, of lipid
origin) were observed, but the fraction of these cells was
considerably reduced compared to the number of these
cells in the previous experiment (Table 1). The cyto-
plasm of hepatocytes in most cases was loose and mostly
crumbly, with several large vacuoles, and their size was
moderately increased.
Electron micrograph of liver cells. In sinusoid lumen a large liver
macrophage with foamy cytoplasm, filled by numerous electron light
Denotes: Mph–macrophage; Hep–hepatocyte; S-sinusoid
*-electron light granules of macrophages
Poloxamer was administered i.p. as a single dose (1000 mg/kg).
Study was performed at the 1st day after administration of a single dose
of poloxamer 407.
Figure 3. Influence of poloxamer 407 on ultrastructure of liver
3.2. Effect of Poloxamer 407 on Fractional
and Subfractional Composition of
Serum lipoproteins
3.2.1. Lipoprotein-Cholesterol (C-LP)
Compared to intact (Control) mice, there was a signifi-
cant (6-fold) increase in the serum total C-LP at 24 and
48 hrs, as well as day 4, in mice which were adminis-
tered poloxamer 407 (Table 2). As shown in Table 2,
among the total C-LP, the greatest increase (50-fold)
was observed with the pro-atherogenic C-VLDL fraction
(both C-VLDL1-2 and C-VLDL3-5 subfractions), as well
as with intermediate density lipoproteins (C-IDL) (20-
fold). However, it should be noted that serum concentra-
tions of each fraction begin to decrease by day 4. The
C-LDL concentration increased about twice, relative to
controls, and continued to remain elevated at day 4.
C-LDL1-3 was moderately (3- to 5-fold) increased at 48
hrs and at day 4 (Table 2). The C-IDL and TG-IDL sub-
fractions were increased significantly (20-30-fold),
while the C-LDL1-3 subfraction was moderately in-
creased (3- to 5-fold) at 48 hrs and at day 4.
Among anti-atherogenic fractions and subfractions,
only a moderate increase of C-HDL was noted (2- to
T. A. Korolenko et al. / HEALTH 2 (2010) 722-730
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Table 2. Concentration of C-LP fractions and subfractions (mg/dl) in murine serum during administration of poloxamer 407 (1000
LP Control, intact (n = 10) Poloxamer, 24 hrs (n = 10) Poloxamer, 48 hrs (n = 8) Poloxamer, 4 days (n = 5)
C-LP (All) 107.4 ± 8.84 662.9 ± 82.40*** 599.7 ± 104.11** 727.2 ± 87.2*
C-VLDL 6.5 ± 1.85 345.8 ± 46.90*** 271.3 ± 73.02** 134.4 ± 46.23**
C-VLDL1-2 0.3 ± 0.07 16.7 ± 2.13*** 12.8 ± 4.70** 8.9 ± 4.11
C-VLDL3-5 6.0 ± 1.71 329.1 ± 45.54*** 258.5 ± 70.44** 125.5 ± 42.43**
C-LDL 43.1 ± 7.67 106.9 ± 24.15** 201.0 ± 34.38*** 247.3 ± 31.52***
C-IDL 1.9 ± 1.61 40.3 ± 11.13** 62.6 ± 14.88*** 34.9 ± 13.32*
C-LDL1-3 41.2 ± 8.61 66.6 ± 25.54 146.1 ± 32.68** 212.4 ± 35.56**
C-HDL 57.9 ± 9.75 210.2 ± 48.36** 127.4 ± 41.93 45.4 ± 13.30
C-HDL2 43.1 ± 10.02 166.5 ± 38.70** 91.9 ± 47.31 16.9 ± 8.55
C-HDL3 14.7 ± 3.55 43.7 ± 20.21 35.5 ± 8.94 * 28.5 ± 9.23
The data are shown as mean ± SEM (*p< 0.05; **p < 0.01; ***р < 0.001 vs control)
The number of animals is in the parentheses.
Abbreviations: C-LP–cholesterol of lipoproteins; C-HDL–cholesterol of high density lipoproteins; C-LDL–cholesterol of low density lipoproteins;
C-IDL–cholesterol of intermediate density lipoproteins; C-VLDL–cholesterol of very low density lipoproteins.
4-fold), with a return to baseline by day 4 (primarily due
to a decrease in the concentration of the C-HDL2 sub-
fraction), while the C-HDL3 subfraction showed a mod-
erate increase (Table 2).
3.2.2. Lipoproteins-TG (TG-LP)
In general, the total amount of TG-LP increased more
significantly as compared to C-LP (20-fold), with a
tendency to return to normal levels by day 4 (Tables 2
and 3). TG-VLDL “behavior” was similar to C-VLDL
(increased 20- to 40-fold), and both subfractions (TG-
VLDL1-2 and TG-VLDL3-5) were responsible for the
elevation of the TG-VLDL fraction (Tables 2 and 3).
Similar to C-LDL, an increase in TG-LDL was observed
(6- to 8-fold). The level of C-IDL (Table 2) and
TG-IDL (Table 3) was shown to be increased (~18- to
28-fold, respectively) with a significant difference at 24,
48 hrs, and on day 4, as compared to the controls (Ta-
bles 2 and 3). A moderate elevation was also observed
for C-HDL and TG-HDL (2- to 3-fold) by 24 hrs, but
serum levels were not significantly greater than controls
from 24 hrs onward (Tables 2 and 3). The TG-HDL2
subfraction was elevated only at 24 hrs after poloxamer
407 administration when compared to controls (Table 3).
There were no significant changes of the TG-HDL3 sub-
fraction (Table 3).
Atherosclerosis is a complex disorder of lipid metabo-
lism and a chronic inflammatory disease [16]. Changes
in lipoprotein metabolism and hyperlipidemia play an
important role in the progression of atherosclerosis. The
initial period of atherosclerosis development and the
interaction of lipoproteins with cells involved in the dis-
lipidemic state merits further investigation. Mouse mod-
els of hyperlipidemia and atherosclerosis are useful tools
with which to study the role of “individual” lipoproteins
of different classes.
Lipoproteins are divided into 4 main fractions (classes)
—HDL, LDL, VLDL, and chylomicrons, or 7 subfrac-
tions (HDL3, HDL2, LDL, IDL, VLDL3-5, VLDL1-2, chy-
lomicrons), or 16 sub/subfractions [15,17,18]. Most se-
rum cholesterol, about 60-70% of total serum cholesterol,
is known to be contained in LDL-C as the major athero-
genic lipoprotein fraction [1]. During the last decade,
several new methods, including the analytical ultracen-
trifugal micromethod, were developed for identification
of atherogenic LDL, and their subclasses, without their
preparative isolation [19].
At the same time, it is important to investigate other
lipoprotein fractions—namely, VLDL and HDL, as well
as their subfractions. VLDL, which is a precursor of LDL,
and some forms of VLDL, is atherogenic, especially
their remnant forms. VLDL contains most of the serum
triglycerides, their role, of which, is now thought to be
an independent and important risk factor in atherosclero-
sis [20]. HDL is known as an antiatherogenic fraction of
lipoproteins [21]. With the assistance of the new physico-
chemical SAXS method, it is possible to simultaneously
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Table 3. Concentration of TG-LP fractions and subfractions (mg/dl) in murine serum during administration of poloxamer 407 (1000
LP fractions, Subfractions Control, intact (n = 10) Poloxamer, 24 hrs (n = 10)Poloxamer, 48 hrs (n = 8) Poloxamer, 4 days (n = 5)
TG-LP (All) 54.5 ± 6.57 1107.4 ± 148.48*** 902.2 ± 223.69** 517.3 ± 152.67**
TG-VLDL 19.5 ± 4.85 952.1 ± 129.69*** 735.2 ± 198.61** 383.0 ± 140.44**
TG-VLDL1-2 3.1 ± 0.61 148.7 ± 26.59*** 103.9 ± 36.43** 80.1 ± 41.67
TG-VLDL3-5 16.0 ± 4.30 803.4 ± 114.37*** 631.3 ± 175.49** 302.9 ± 101.23**
TG-LDL 14.0 ± 1.85 80.7 ± 13.92** 120.7 ± 33.23** 117.8 ± 14.79***
TG-IDL 2.6 ± 2.15 53.8 ± 14.83** 83.4 ± 19.82** 46.6 ± 17.74*
TG-LDL1-3 11.4 ± 2.31 24.6 ± 10.37 47.5 ± 11.14** 71.2 ± 15.19**
TG-HDL 21.2 ± 3.58 76.9 ± 17.67** 46.4 ± 15.21 16.5 ± 5.00
TG-HDL2 15.9 ± 3.68 60.9 ± 14.31** 33.6 ± 17.17 6.4 ± 3.16
TG-HDL3 5.3 ± 1.25 16.1 ± 7.38 12.8 ± 3.14 10.14 ± 3.39
The data are shown as mean ± SEM (*p < 0.05; **p < 0.01; ***р < 0.001 vs control)
The number of animals is in the parentheses.
Abbreviations: TG-LP-triglycerides of lipoproteins; TG-HDL–triglycerides of high density lipoproteins; TG-LDL–triglycerides of low density lipo-
proteins; TG-IDL–triglycerides of intermediate density lipoproteins; TG-VLDL–triglycerides of very low density lipoproteins.
investigate all three major classes of lipoproteins (VLDL,
LDL, HDL) and all subfractions, except chylomicrons
(the particles are too large for this method of analysis)
[4]. Moreover, it is known that fasting human plasma, as
well as plasma of mice 12-14 hrs after feeding, contains
negligible chylomicrons. The available physical-chemical
data in the literature suggests the existence of a stable
thermodynamic equilibrium between all lipoprotein types.
This equilibrium is specific for each physiological state
in the human and helps to stabilize the lipoprotein struc-
ture and assist with normal lipoprotein functioning. In
the SAXS method, the dynamic equilibrium between
different LP types was used as a physico-chemical basis,
and a general model, which takes into account the rela-
tive amount of all LP types and their transitional forms
(in terms of the relative size of LP particles and their
relative concentrations) was developed [4].
As was shown in this work, the poloxamer 407-induced
model of hyperlipidemia in mice was characterized by a
significant elevation of both C-LP and TG-LP total frac-
tions. Similar data were obtained by other investigators
that studied the level of atherogenic and antiatherogenic
lipoprotein fractions with other, more common methods
[5]. It is necessary to mention that the concentration of
pro-atherogenic LDL and VLDL fractions increased dra-
matically, as compared to a moderate increase in the
level of HDL particles. This is probably one of the rea-
sons why repeated administration of poloxamer 407 to
mice successfully reproduces the atherosclerotic process
in rodents, where atherosclerosis is difficult to develop
due to the normally high concentration of antiathero-
genic HDL. These data were supported by the observa-
tion that statin treatment in patients with cardiovascular
diseases decreases the mortality by decreasing the C-LDL
levels, thereby, exerting the protective lipid-lowering and
also pleiotropic effects [2].
Elevated plasma concentration of C-LDL is consid-
ered a risk factor for atherosclerosis development. LDL,
especially oxLDL particles, infiltrates the vascular wall
and can be taken up by macrophages. This is followed
by secretion of proinflammatory factors which are in-
volved in the pathogenesis of atherosclerosis. HDL, like
other fractions of lipoproteins (LDL and VLDL), are a
heterogeneous group of lipoproteins; their core contains
some free cholesterol, triglycerides, and cholesterol es-
ters [21,22]. HDL particles are involved in reverse cho-
lesterol transport, and their atheroprotective role may be
connected with regulation of adhesion molecule expres-
sion and the prevention of oxidative modifications of
LDL [16,21]. It was shown in this work that both sub-
fractions of C-HDL were only moderately increased in
poloxamer 407-treated mice and returned to normal lev-
els by day 4 following poloxamer administration. Simi-
lar changes were observed in the TG-HDL fraction and
In general, there are many factors influencing serum
T. A. Korolenko et al. / HEALTH 2 (2010) 722-730
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
concentration of lipoproteins (biosynthesis, uptake by
endocytosis, secretion, and degradation). The liver is the
main source of VLDL. LDL catabolism occurs primarily
in the liver and in most extrahepatic tissues [2]. Different
types of cells have been shown to have LDL receptors
on the plasma membrane [6]. Catabolism of LDL has
been shown to occur inside of lysosomes [23,24]. Me-
tabolism of HDL is closely related to homeostasis of
lipids in the intact vertebrate, with lipoproteins residing
in both the serum and in tissues [2].
Poloxamer 407 appears to act as a general lipase in-
hibitor. Mammalian lipases include three main lipase
members; namely, lipoprotein lipase, hepatic lipase, and
endothelial lipase, acting in vitro and in vivo on lipopro-
teins [23]. Lipoprotein lipase has a high triglyceride li-
pase activity, while endothelial lipase exerts phospholi-
pase activity. Endothelial lipase is able to hydrolyze lip-
ids from HDL, while hepatic lipase exerts functional
activity on all classes of lipoproteins [23]. Lipoprotein
lipase (EC (LPL) is a key enzyme in lipopro-
tein metabolism. LPL belongs to the class of enzymes
known as hydrolases, which catalyze the degradation of
glycerides, including triglycerides. Poloxamer 407 (as
well as the nonionic detergent Triton WR 1339) has been
shown to inhibit LPL, and this effect has been suggested
as playing a crucial role in disturbances of lipoprotein
transport and a significant elevation in the serum levels
of LP-TG.
There are some similarities in the models of polox-
amer and Triton WR 1339-induced hyperlipidemia fol-
lowing a single intraperitoneal administration of each
agent. Both models were characterized by dramatic in-
creased serum concentrations of cholesterol and, espe-
cially triglycerides, in rats and mice. As mentioned
above, the resultant hypertriglyceridemia occurs due to
surfactant-mediated inhibition of LPL’s enzymatic activ-
ity [25,26]. Moreover, Triton WR 1339 is a well-known
lysosomotropic agent, accumulating inside of lysosomes
of liver cells; specifically, hepatocytes and Kupffer cells
[27]. In both species of rodents, Triton-induced hyper-
lipidemia occurred 24 hrs after detergent administration,
and was followed by a dramatic increase in both lipo-
protein cholesterol and, especially, of lipoprotein triglyc-
eride concentrations [26,27]. We have recently shown
significant increases in the concentration of atherogenic
C-LDL, C-VLDL (due to an increase of the VLDL3-5
subfraction), and IDL in mice, and even more profound
elevations in rats (Korolenko et al., submitted In Press).
These hyperlipidemic animal models can be used to
study the role of lipoproteins, especially lipoprotein
triglycerides, (but also C-LDL and C-VLDL) in the
pathogenesis of atherosclerosis, as well as for testing the
efficacy of new hypolipidemic drugs. The poloxamer
407-induced mouse model of hyperlipidemia would ap-
pear to resemble a Type 2a/2b or 3 dislipidemia in hu-
Mice and rat models of hyperlipidemia are used more
often now because of their convenience, the ability to
genetically alter the animals to evaluate the effect of a
particular gene on lipid metabolism, and because they
are cost-effective. The poloxamer 407-induced mouse
model of dyslipidemia and atherosclerosis reliably re-
produces the hyperlipidemic state, and, if administered
on a repetitive basis (or, if dosed continuously with the
aid of an implanted osmotic pump), causes formation of
fibrofatty aortic lesions in the same size and number as
classic diet-induced or gene-knockout mouse models [28].
Poloxamer 407 has been used in several fields of medi-
cine as a nano-carrier for different drugs for the treat-
ment of several inflammatory diseases and tumors [29].
However, when used to induce dyslipidemia and athero-
sclerosis in C57BL/6 mice, it must be emphasized that
these are supraphysiologic doses of P-407 that would
never be utilized in any commercial formulation em-
ploying its sustained-release or reverse-thermal gelation
properties. Additionally, most applications of poloxamer
407 in the pharmaceutical literature deal with non-par-
enteral routes of delivery, unlike the intraperitoneal ad-
ministration of poloxamer 407 to intentionally induce
dyslipidemia in mice [30-34].
In conclusion, we have identified changes in the lipid
fractions and subfractions following a single dose of
poloxamer 407 to mice, as well as characterized the rela-
tive spleen and liver weights, and the effects on blood
leukocytes. Additionally, we have pioneered the use of
SAXS as a method to determine the concentrations of
the serum lipids and lipid subfractions. This new ana-
lytical approach can now be successfully employed to
determine the concentrations of lipoprotein fractions,
and their subfractions, in either mouse or human plasma
samples for a more complete picture of the hyperlipi-
demic state.
This work was partially supported by integrated grant of Siberian
Branch of the Russian Academy of Medical Sciences and Far East
Division of Russian Academy of Sciences on natural immunomodula-
tors study (2006-2008). Authors are grateful to engineer M.Tuzikov for
help in Small-angle X-ray scattering, Dr. Monoszon A., Dr. Kisarova
Ya., Dr. Sukhovershin R., Dr. K. Loktev for technical help during
preparing of the manuscript.
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C (TG)-HDL–cholesterol (triglycerides) of high density
C (TG)-LDL–cholesterol (triglycerides) of low density
C (TG)-IDL–cholesterol (triglycerides) of intermediate
density lipoproteins
C (TG)-VLDL–cholesterol (triglycerides) of very low
density lipoproteins
SAXS-Small-angle X-ray scattering