CVR-2012-726R1
Supplementary Data
Supplementary methods
Animals and diets
Thirty 8-weeks old male Golden Syrian hamsters weighing 113.24 ± 7.4 g were randomly
assigned to two groups (n = 15 for each group). They were maintained in a 12:12 (light/dark)
cycle at 22 ± 2 ºC and 50 ± 10 % relative humidity with free access to both food and water.
Food intake and body weight were recorded every week. The control group ate a diet based
on Harlan Tecklad 7001 (Harlan Laboratories, Barcelona, Spain) composition and the
atherogenic group ate an atherogenic diet 1 in which the cholesterol content had been set at
0.5% and which was supplemented with 15% of lard (Table 1 of the Supporting Information).
These diets were maintained for 3 months. Animals were then fasted overnight, anesthetised
with 2.5% isoflurane in air and finally sacrificed by cardiac puncture obtaining the blood which
was immediately centrifuged at 2000 x g for 5 min for plasma collection. Then, aorta was
removed and separated the aortic arcs from the descendent aorta. The descendent aortas
were frozen in liquid N2 and stored at -80 ºC until analysis. The aortic arcs were put in
paraformaldehyde 10% during 24 h at 4ºC, dried and frozen and stored at -80 ºC until analysis.
Later, transverse sections (16 µm) from arch of aorta were obtained and collected on gelatincoated glass slices.
In order to analyse dietary modulation of atheromatosis, an additional group was introduced
(n = 15). The grape seed extract (961016, Euromed S.A. Barcelona, Spain) supplemented group
(GSE) ate the same diet of the atherogenic group but supplemented with a 0.2% of GSE extract
(Table 1 of the Supporting Information).
All experimental procedures were approved by the Institutional Animal Care Committee of
IRBLleida and were conformed with the Directive 2010/63/EU of the European Parliament.
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Lipidomics analysis
For aorta samples, 50 µl of cold methanol containing representative internal standards 2 were
added to 7-12 mg of tissue and homogenized with a Potter– Eljeveim device at 4ºC. Then, 500
µl of chloroform and 187.5 µl of 0.7% of KCl were added. It is necessary to vortex after each
solvent addition. Then, the samples were vortexed and centrifuged at 1000 x g, 4 ºC for 10
min, and the chloroform phase was separated in a glass tube. Five hundred µl of chloroform
were added to the sample and the centrifugation step was repeated. Finally, the chloroform
phase (both from first and second extraction) was evaporated using a Speed Vac (Thermo
Fisher Scientific, Barcelona, Spain) and resuspended in chloroform:methanol (1:3, v/v).
For plasma samples, 175 µl of phosphate buffer were added to 25 µl of plasma. Then, 600 µl of
cold acetone containing lipid class representative internal standards were added 2, vortexed
for 10 s, incubated at 4ºC for 30 min and centrifuged at 1000 x g at 4 ºC for 10 min, to
precipitate proteins. Supernatants were extracted and 250 µl of methanol, 500 µl of
chloroform and 200 µl of 0.7% of KCl were added. It is necessary to vortex after each solvent
addition. Then, the samples were vortexed and centrifuged at 1000 x g, 4 ºC for 10 min, and
the chloroform phase was separated in a glass tube. Five hundred µl of chloroform were added
to the sample and the centrifugation step was repeated. Finally, the chloroform phase (both
from first and second extraction) was evaporated using a Speed Vac (Thermo Fisher Scientific,
Barcelona, Spain) and resuspended in chloroform:methanol (1:3, v/v).
For LC-Q-TOF-based lipid molecular species analyses, lipid extracts were subjected to massspectrometry using a HPLC 1200 series coupled to ESI-Q-TOF MS/MS 6520 (Agilent
Technologies, Barcelona, Spain) . One μl of sample was applied onto a reverse phase column
(Luna C5, 3.5 µm, 4.6x50 mm, Phenomenex, LA; CA, USA) equipped with a reverse phase
precolumn (C4, 3.5 µm, 2x20 mm, Phenomenex, LA; CA, USA) for positive ionization, and onto
a reverse phase column (Gemini C18, 3.5 µm, 4.6x50 mm, Phenomenex, LA; CA, USA) equipped
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with a reverse phase precolumn (C18, 3.5 µm, 2x20 mm, Phenomenex, LA, CA, USA) for
negative ionization. The flow rate was 200 μl/min with solvent A composed of 95% water, 5%
methanol containing 0.1% formic acid and 5mM ammonium formate for positive ionization or
0.1% ammonium hydroxide for
negative ionization, and solvent B composed of 65%
isopropanol, 30% methanol, 5% water containing corresponding counterions. The gradient
consisted of solvent B from 0% to 20% in 5 minutes, from 20% to 100% in 60 minutes, return to
0% B in 20 minutes, and re-equilibrated at 0% solvent B for 10 min.
Data were collected in positive and negative electrospray ionization mode TOF operated in fullscan mode at 100 to 3000 m/z in an extended dynamic range (2 GHz), using N2 as nebulizer gas
(5 L/min, 350ºC). The capillary voltage was 3500 V with a scan rate of 1 scan/s. The ESI source
used a separate nebulizer for the continuous, low-level (10 L/min) introduction of reference
mass compounds: 121.050873, 922.009798 (positive ion mode) and 119.036320, 966.000725
(negative ion mode), which were used for continuous, online mass calibration. The
MassHunter Data Analysis Software (Agilent Technologies, Barcelona, Spain) was used to
collect the results and the MassHunter Qualitative Analysis Software (Agilent Technologies,
Barcelona, Spain) to obtain the molecular features of the samples, representing different, comigrating ionic species of a given molecular entity using the Molecular Feature Extractor
algorithm (Agilent Technologies, Barcelona, Spain), as described previously
3, 4
. Finally, the
MassHunter Mass Profiler Professional Software (Agilent Technologies, Barcelona, Spain) was
used to perform a non-targeted lipidomic analysis over the extracted features. Only common
features (found in at least 75% of the samples of the same condition) were taken into account
to correct for individual bias. Multivariate and clustering analysis were obtained using this
software. The masses representing significant differences by Student T-test (fold change ≥ 2, p
< 0.05 with Benjamini-Hochberg Multiple Testing Correction) were searched against the LIPID
MAPS
5
database. The identities obtained were then compared to the authentic standards
added.
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Targeted lipidomic analysis was performed using the same chromatographic and
spectrometric method as untargeted approach. MassHunter Qualitative Analysis Software
(Agilent Technologies, Barcelona, Spain) was employed for integration and extraction of peak
intensities of the different lipid species. We searched for a) different free fatty acids as
docosahexaenoic acid, oleic acid, linolenic acid, stearic acid, arachidonic acid, lauric acid,
palmitic acid, capric acid and myristic acid, b) cholesterol and c) different soluble oxidative
stress-related markers as 10-hydroxy-docosahexaenoic, 17-hydroxy-docosahexaenoic, 8isoprostaglandin F2α, 13-hydroxyoctadecadienoic acid (HODE), 9-HODE, HODE-cholesteryl
ester,
15-hydroxyeicosatetraenoic
phosphatidylcholine
(PGPC),
acid
(HETE),
1-palmitoyl-2-glutaryl-sn-glycero-3-
1-O-hexadecanoyl-2-O-(9-carboxyoctanoyl)-sn-glycero-3-
phosphatidylcholine (PazPC), 4-hydroxynonenal, 10-nitrooleate, cholesterol-5α,6α-epoxide, 5cholesten-3β-ol-7-one,
7β-hydroxycholesterol,
resolvin
D1,
cholesteryl
linoleate
hydroperoxide, and cholesteryl linoleate.
The m/z values used for quantification of lipid molecules detected were: m/z 245.2486 [M-H]for oleic acid, m/z 283.2643 [M-H]- for stearic acid, m/z 255.233 [M-H]- for palmitic acid, m/z
279.233 [M-H]- for linoleic acid, m/z 303.233 [M-H]- for arachidonic acid, m/z 327.233 [M-H]for docosahexaenoic acid, m/z 369.3487 [M+H]++[-H2O] for cholesterol; m/z 401.3414 [M+H]+
for 5-cholesten-3β-ol-7-one and for 7β-ketocholesterol, m/z 403.3571 [M+H]+ for cholesterol5α,6α-epoxide , m/z 321.2424 [M+H]+ for HETE, m/z 295.2318 [M+H]+ 9, 13-HODE and m/z
610.3715 [M+H]+ for PGPC.
Metabolomic analyses
Thirty μl of cold methanol was added to 10 μl of plasma, vortexed for 1 min, and incubated at
−20°C for 1 h to precipitate proteins. Samples were centrifuged at 12000 x g for 3 min, and the
supernatant was collected. The supernatant was dried in a SpeedVac and resuspended in 50 μl
of water. The sample was filtered in an eppendorf UltraFree 5 kDa filter.
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Four microliters of extracted sample were applied to a reverse-phase column (C18 Luna 3 n
pfp(2) 100 A 150 × 2 mm, Phenomenex, Torrence, CA) equipped with a precolumn (AJO-8326
pfp(2) 4 × 2 mm, Phenomenex, Torrence, CA). The flow rate was 200 μL/min. Solvent A was
composed of water containing 0.1% formic acid for positive ionization or 0.1% acetic acid for
negative ionization, and solvent B was composed of 95% acetonitrile and 5% water containing
corresponding counterions. The gradient consisted of solvent B from 5% to 100% over 20 min,
held at 100% solvent B for 5 min, and re-equilibrated at 5% solvent B for 6 min.
Data were collected in positive and negative electrospray mode TOF operated in full-scan
mode at 100–3000 m/z in an extended dynamic range (2 GHz), using N2 as the nebulizer gas (5
L/min, 350 °C). The capillary voltage was 3500 V with a scan rate of 1 scan/s. The ESI source
used a separate nebulizer for the continuous, low-level (10 L/min) introduction of reference
mass compounds: 121.050873, 922.009798 (positive ion mode) and 119.036320, 966.000725
(negative ion mode), which were used for continuous, online mass calibration. The Masshunter
Data Analysis Software (Agilent Technologies, Barcelona, Spain) was used to collect the results,
and the Masshunter Qualitative Analysis Software (Agilent Technologies, Barcelona, Spain) was
used to obtain the molecular features of the samples, representing different, comigrating ionic
species of a given molecular entity using the Molecular Feature Extractor (MFE) algorithm
(Agilent Technologies, Barcelona, Spain), as described 3, 4. Finally, the Masshunter Mass Profiler
Professional Software (Agilent Technologies, Barcelona, Spain) was used to perform a
nontargeted metabolomic analysis of the extracted features. Only common features (found in
at least 75% of the samples of the same condition) were analysed, correcting for individual
bias. The masses with significant differences in abundance (determined using a Student’s t
test; fold change ≥ 2, p < 0.05) were searched against the various databases (METLIN 6, HMDB
7
, LIPID MAPS 5, and KEGG 8 databases), using exact masses.
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CVR-2012-726R1
To confirm the identity of the metabolites obtained after the nontargeted analysis (MS analysis
and database search), we used the Q-TOF mode operated in full scan mode (MS and MS/MS)
at 100–1500 m/z. The capillary voltage was 3500 V with a scan rate of 3 (MS) or 5 (MS/MS)
scans/s with a fixed collision energy (20 V). N2 was used as a gas nebulizer (5 L/min flow rate,
350 °C).
Angiotensin conversor enzyme (ACE) activity
ACE activity in serum was determined following a fluorescence-based method previously
described 9. Briefly, 25 µL of serum was mixed with 25 µL of 150 mM Tris-base buffer (pH 8.3)
and 100 µL of ACE fluorescent substrate (0.45 mM of Abz-Gly-Phe(NO2)-Pro) in a 96-well
microplate. Appropriate controls (25 µL of serum + 125 µL of Tris-base buffer) were included in
separate wells. After 10 min of incubation at 37 ºC, kinetic fluorescence (Ex/Em 355/400)
readings were measured during 30 min (1 reading each 5 min) using a Tecan Infinite 11200.
ACE activity was determined as the slope of the linear regression obtained from the kinetic
measure. ACE activity of samples was compared with the control group.
Aorta immunohistochemistry
For immunofluorescence in aorta samples, the slices were permeabilized with 0.1% Triton X100 and blocked with 20% of normal horse serum in 0.1% Triton X-100 for 2h. Then, the slices
were incubated at 4 °C for 24 h either with: (1) the rabbit anti-CD36 polyclonal antibody
(ab78054) (diluted 1:250, Abcam, Cambridge, UK) and (2) the mouse anti-PDI-ER Marker
monoclonal antibody (ab2792) (diluted 1:250, Abcam, Cambridge, UK). The slices were
incubated at room temperature for 1h with the appropriate secondary antibodies: Alexa Fluor
488 goat anti-mouse (diluted 1:750, Molecular Probes, Eugene, OR, USA) or/and Alexa Fluor
546 goat anti-rabbit (diluted 1:750, Molecular Probes, Eugene, OR, USA). Slices were finally
counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI, 50 ng/ml) for 5 min
and mounted on slides with Vectashield (Vector Laboratories, Burlingame, CA, USA). Mounted
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CVR-2012-726R1
slices were examined under a Fluoview 500 Olympus confocal laser scanning microscope
(Olympus, Hamburg, Germany).
Cell culture experiments
PPARγ transcriptional activity response in human embryonic kidney cells (HEK293)
For gene expression, Reporter Gene methodology was employed. HEK293 cells were grown in
Advanced MEM (Invitrogen Co., Carlsbad, CA) supplemented with 10% fetal bovine serum at
37ºC. P1015 (PPRE X3-Tk-luc) and P20702 (pHM829, LacZ-MCS-GFP) plasmids were employed
for PPARγ transcriptional activation analysis and control of transfection, respectively
10, 11
.
Transient transfection of two plasmids was performed using LipofectAMINE Plus according to
the manufacturer’s instructions (Invitrogen Co., Carlsbad, CA). Plasmids were purchased from
Addgene (www.addgene.org). Plasmids amplification was performed in E. coli and then
isolated with a Plasmid Maxi Prep (Qiagen Iberia SL, Madrid, Spain), according to manufacturer
instructions. Later, HEK293 cells at a density of 106 cells/plate were transfected with 20 µg of
PPRE X3-Tk-luc along with 10 µg of pHM829. After incubation for an additional 24 h, the cells
were harvested and control of transfection was measured by GFP fluorescence. Further, cells
were trypsinized and harvested for 24 h in a 96 well plate. Then, depending on the experiment
performed, cells were exposed to: a) plasma of control, atherogenic and GSE groups at a dose
of 10% v/v with Advanced MEM; b) 5, 20, 100, 200, 400, 1000, 2000 and 5000 nM of
taurocholic acid (Sigma Aldrich, S. Louis, MO, USA) or c) 5, 20, 100, 200, 400 and 1000 mg/L of
GSE extract and incubated during 5 h. Additionally, in separate wells, as a control of increased
PPARγ activation, a calibration curve with Bezafibrate (Sigma Aldrich, S. Louis, MO, USA) from
1 M to 200 M was employed. To determine gene expression a Nova-Bright β-galactosidase
and Firefly Luciferase Dual Enzyme Reporter Gene Chemiluminiscent Detection System
(Invitrogen, Calsbarg, CA, USA) was employed. The activation of PPARγ was measured as the
intensity of luminescence after the exposure of Firefly Luciferase up to 45 min and normalized
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by β-galactosidase activity both measured according manufacturer instructions in an Infinite
200 Pro instrument (Tecan Group Ltd, Männedorf, Switzerland).
DNA double strand break induction in human microvascular endothelial cell (HMEC)
HMEC cell line was grown in DMEM medium (LONZA) supplemented with 10% fetal bovine
serum heat inactivated (Invitrogen), 2 mM L-Glutamine (Invitrogen Co., Carlsbad, CA) and 20
U/mL penicilline and 20 µg/mL streptomicine (Invitrogen Co., Carlsbad, CA) as antibiotics.
Then, depending on the experiment, cells were exposed to a) plasma of control, atherogenic
and GSE groups at a dose of 10% v/v with DMEM serum free; b) 5, 100, 200 and 1000 nM of
taurocholic acid (Sigma Aldrich, S. Louis, MO, USA) or c) 200 and 800 mg/L of GSE and
incubated during 8 h. For evaluating preventive senescence effect of the compounds, the cells
were subsequently incubated with the DNA alkylating agent methyl methanesulfonate (MMS)
(Sigma Aldrich, S. Louis, MO, USA) at 1.5 mM for 6 hours.
For immunofluorescence assay, cells were fixed for 1 h 4°C with 4% paraformaldehyde in PBS
and permeabilized with 0.1% Triton X-100 in PBS for 30 min. After blocking with 10 % Normal
Horse Serum (0.1 % Triton in PBS) for 1 hour, cells were incubated at 4° C with anti-gamma
H2AX (phospho S139) antibody (ab2893) diluted 1:750. Antigen was detected with secondary
antibody conjugated to Alexa-Fluor-546 diluted 1:750. Cells were finally counterstained with
DAPI (50 ng/ml) (Vector Laboratories, Burlingame, CA, USA), coverslipped using Vectasheild
mounting media and examined under a Fluoview 500 Olympus confocal laser scanning
microscope (Olympus, Hamburg, Germany).
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Supplementary results
Correlational analysis
The correlation analyses (using the samples of the three groups) revealed several significant
correlations (Supplementary Figure 3A). Specifically, the analyses revealed positive
correlations between 7-ketocholesterol in plasma with total, LDL and HDL cholesterol (Pearson
Correlation = 0,632, p = 0,004; Pearson Correlation = 0,487, p = 0,049; Pearson Correlation =
0,575, p = 0,001, respectively). The 5α,6α-epoxy-cholesterol in plasma positively correlate with
the levels of palmitic, stearic and oleic acid in plasma (Pearson Correlation = 0,329, p = 0,038;
Pearson Correlation = 0,406, p = 0,009; Pearson Correlation = 0,434, p = 0,005, respectively).
Further, we found positive correlation between HETE and arachidonic acid in aorta (Pearson
Correlation = 0,569, p = 0,014), indicating that the level of arachidonic acid (the native free
fatty acid) is the principal reason for its oxidation and, therefore, for HETE formation. Contrary,
HODE aorta levels did not correlate with its precursor, the linolenic acid, whereas we found
high positive correlation with two other free fatty acids in aorta, the stearic acid (Pearson
Correlation = 0,837, p = 0,000) and the oleic acid (Pearson Correlation = 0,810, p = 0,000). The
levels of total and LDL cholesterol in plasma also positively correlate with the levels of DHA in
aorta (Pearson Correlation = 0,542, p = 0,030; Pearson Correlation = 0,540, p = 0,031,
respectively).
Concerning metabolites found in plasma, the correlational analysis showed that both taurine
and phenylalanine negatively correlate with total and LDL-cholesterol (phenylalanine-total
cholesterol: Pearson Correlation = 0,672, p = 0,000; phenylalanine-LDL-cholesterol: Pearson
Correlation = 0,637, p = 0,000; taurine-total cholesterol: Pearson Correlation = 0,663, p =
0,000; taurine-LDL-cholesterol: Pearson Correlation = 0,601, p = 0,000), being the correlation
between these two metabolites positive and almost perfect (Pearson Correlation = 0,936, p =
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0,000). This last result suggested direct regulation between these metabolites. Finally, the
levels of the taurocholic acid in plasma positively correlate with the plasma levels of total
cholesterol, LDL-cholesterol, oleic acid, arachidonic acid and DHA (Pearson Correlation = 0,502,
p = 0,004; Pearson Correlation = 0,515, p = 0,003; Pearson Correlation = 0,349, p = 0,034;
Pearson Correlation = 0,407, p = 0,012; Pearson Correlation = 0,407, p = 0,012, p = 0,000,
respectively). Most interesting, the levels taurocholic acid positively correlates with the levels
of free cholesterol in aorta (Pearson Correlation = 0,700, p = 0,004), being a potential good
biomarker of atheromatosis process (Figure 2D).
Further, if we performed the correlational analysis for each group (Supplementary Figure 3B, C
and D) we could see that the diet affects the correlations between molecules suggesting
different regulation of lipid and metabolites metabolism.
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Supplementary Figures
Supplementary Figure 1
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Figure S1. Metabolomic (A and B) and lipidomic (C and D) analyses in plasma samples in the
preliminary GSE intake in the non-atherogenic context. A and C. Heat map representation of
hierarchical clustering of molecular features (see main text for definition) found in each
sample of two groups (CTL: control group, GSE: Grape seed extract supplemented group). Each
line of this graphic represents an accurate mass ordered by retention time, coloured by its
abundance intensity normalized to internal standard and baselining to median/mean across
the samples. The scale from -10.8 (blue) to 10.8 (red) represents this normalized abundance in
arbitrary units. B and D. Tridimensional PCA and PLS-DA graphs demonstrated the effect of
GSE in the plasma metabolome and lipidome. Control animals were represented in red spots
and GSE animals in blue spots.
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Supplementary Figure 2
Figure S2. Sphingolipid metabolism based on KEGG pathway database 8
.
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Supplementary Figure 3
Figure S3. Correlation analysis between lipid and molecules involved in atherogenic processes
using samples form three groups (A), control (ctl) group (B), atherogenic (Ath) group (C) and
grape seed extract (GSE) group (D).
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Supplementary Figure 4
Figure S4. Taurocholic acid (TA)induces double strand breaks in DNA in endothelial cells in
culture (HMEC), as evidenced by confocal microscopy after immunocytochemistry with antiγH2AX antibody. Representative images of 4-6 experiments. Scale bar: 20 µm.
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Supplementary Figure 5
Figure S5. Taurocholic acid (TA) increases methyl methanesulfonate (MMS)-induced double
strand breaks in DNA in endothelial cells in culture (HMEC), as evidenced by confocal
microscopy after immunocytochemistry with anti-γH2AX antibody. Representative images.
Positive Ctl: cells incubated with MMS. Scale bar: 20 µm.
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Supplementary Figure 6
Figure S6. In vivo effects of grape seed extract (GSE) intake are not reproduced by in vitro
incubation of the extract. Double strand breaks in DNA in endothelial cells in culture induced
by methyl methanesulfonate (MMS) are not diminished by GSE coincubation as evidenced by
confocal microscopy after immunocytochemistry with anti-γH2AX antibody. Representative
images from n=4-6 experiments. Positive Ctl: cells incubated with MMS. Scale bar: 20 µm.
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Supplementary Tables
Supplementary Table 1: Diets composition
Component
Control (g)
Atherogenic (g)
GSE (g)
Casein
L-Cysteine
Corn starch
Sugar
Corn oil
Cellulose
Minerals
Vitamins
Lard
Cholesterol
Grape Seed Extract
Total weight
200
3
447
175
80
50
35
10
0
0
0
1000
200
3
393
154
0
50
35
10
150
5
0
1000
200
3
393
154
0
48
35
10
150
5
2
1000
Energy (Kcal/g diet)
Carbohydrates (g/Kg)
Starch %
Sugar %
Energy (Kcal/g diet)
% Energia
Proteins (g/Kg)
Energy (Kcal/g diet)
% Energy
Lipids
Energy (Kcal/g diet)
% Energy
Fiber %
4,02
622
67
26
2,5
62
203
0,8
20
80
0,7
18
7
4,4
547
66
26
2,2
50
203
0,8
18
155
1,4
32
8
4,4
547
66
26
2,2
50
203
0,8
18
155
1,4
32
8
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