Supporting Information
© Wiley-VCH 2008
69451 Weinheim, Germany
Amphiphilic/bipolar metallocorroles that catalyze the
decomposition of reactive oxygen and nitrogen species,
rescue lipoproteins from oxidative damage, and attenuate
atherosclerosis in mice
Adi Haber, Atif Mahammed, Bianca Fuhrman, Nina Volkova, Raymond Coleman,
Tony Hayek, Michael Aviram* and Zeev Gross*
Methods
Chemicals. The corrole metal complexes and peroxynitrite solutions (freshly
prepared on the day of usage) were prepared according to previously described
procedures
reliable
[1, 2]
, while the solvents and standard chemicals were purchased from
sources
and
used
as
received.
This
includes
SIN-1
(3-
Morpholinosydnonimine hydrochloride), EDTA, CuSO4, thiobarbituric acid and folin
reagent, that were purchased from Aldrich. PBS (phosphate buffered saline) was
purchased from Biological industries.
Oxidation of small molecules by peroxynitrite.
a) Formaldehyde from DMSO: 1 mL of an aqueous solution containing NaOH (0.05
M) and peroxynitrite (2.5·10-3 M) was added to a 1 mL phosphate buffer solution that
contained 20 µL DMSO and either no additive or 1-Fe (1.6·10-4 M) or 1-Mn (1.6·10-4
M). The final pH of the solutions was 7.4 and the temperature was maintained at 24
˚C. After 5 min of stirring, a 0.3 mL NaOH solution (7.8 M) was added to each tube
for analyzing the amount of formed formaldehyde. This was immediately followed by
supplying the tubes with 0.3 mL of 34.2 mM purpald in 480 mM HCl; and a second
incubation with continuous shaking was performed for 10 min at 24 °C. The such
obtained reaction product between formaldehyde and purpald was oxidized to a
colored compound by the addition of 0.3 mL 470 mM KIO4 in 470 mM NaOH. The
absorbance at 550 nm was measured, from which the amount of formaldehyde was
determined based on the standard curve obtained from formaldehyde solutions of
known concentrations.
b) Malonyldialdehyde (MDA) from deoxyribose: 10 mM of deoxyribose in buffer
solution was reacted with 390 µM of peroxynitrite in both the absence and the
presence of 0.1 mM 1-Fe or 1-Mn (pH 7.4, T = 24 °C). 1 mL of 2.8% trichloroacetic
acid and 1 mL of 1% thiobarbituric acid in 0.1 M NaOH adjusted to pH 3.5 were
sequentially added to 1 mL samples and the absorbance spectra at 532 nm due to the
absorbance maximum of the MDA-thiobarbiturate product were recorded as a
function of time (0-22 hr) at 24 °C.
c) Nitration of fluorescein: 5 µM of fluorescein in buffer was reacted with 25 µM of
peroxynitrite in both the absence and the presence of 5 µM of 1-Fe or 1-Mn (T = 24
°C, pH 7.4). The changes in fluorescein absorbance were measured on the UV/vis
spectrophotometer. The yield of nitration was determined by converting absorbance to
changes in concentration of fluorescein and nitrated fluorescein, using pre-determined
extinction coefficients. For increased accuracy, the three wavelengths where the
largest changes in absorbance occur were used. The yields were calculated based on
the initial substrate concentration.
d) Nitration of L-tyrosine: Peroxynitrite (0.44 µmol) was reacted with 0.16 µmol of
L-tyrosine in 2 mL phosphate buffer solution, pH 7.4 at T = 24 °C for 5 min. The
concentration of such formed nitrotyrosine was estimated by recording the 438 nm
absorbance (ε = 4200 M-1cm-1) of basified solutions (via the addition of 0.2 ml of 7.8
M NaOH). The same experiments were repeated in the presence of 0.1 µmol 1-Fe or
1-Mn.
e) Oxidation of DMSO by copper sulphate: DMSO (20 µL) was added to 2 mL
phosphate buffer (pH 7.0, T = 24 °C) in both the absence and the presence of 50 µM
1-Fe or 1-Mn. Reaction was initiated by adding a mixture of CuSO4 and
phenanthroline (final concentration of 8 µM for each), followed by sodium ascorbate
(500 µM). After 18 hr of stirring, the amount of produced formaldehyde was assayed
by reaction with purpaldehyde as described above. The same experiment was done
with glutathione instead of sodium ascorbate as reducing agent. The concentrations of
the reagents were: 1 mM glutathione, 32 µM CuSO4, 32 µM phenanthroline, 50 µM
1-Fe, 50 µM 1-Mn.
LDL/HDL preparation. LDL/HDL was separated from plasma of normal healthy
volunteers by sequential ultracentrifugation [3] and dialyzed against saline with EDTA
(1 mM). Protein concentration of the separated fraction was determined with the folin
phenol reagent
[4]
. Before the oxidation study, LDL/HDL was diluted in PBS to 1 g
protein/L and dialyzed against PBS at 4ºC to remove the EDTA.
Interaction of LDL/HDL with corroles. The association between corroles and LDL
was examined by recording the absorbance spectrum of 10 µM aqueous corrole
solutions before and after addition of 100 mg of LDL protein/L (corresponding to a
molar concentration of 0.2 µM). Large changes were induced upon the addition of
LDL, with the most significant being shifts of absorbance maxima from 480 to 475
nm for 1-Mn, from 404 to 410 nm for 1-Fe, and from 424 to 428 nm for 1-Ga. These
solutions ([LDL]/[corrole] = 1/50) were dialyzed extensively against PBS, and the
absorbance after dialysis was compared to that of the pre-treated solutions. The
absorbance decreased by 20, 10 and 30% for 1-Mn, 1-Fe and 1-Ga respectively,
which led to the conclusion that each LDL particle binds 40, 45 and 35 molecules of
1-Mn, 1-Fe, and 1-Ga, respectively, with high affinity. A similar procedure was used
to quantify the interaction of HDL with the corroles, leading to the conclusions that
10 molecules of 1-Ga or 1-Fe and 12 of 1-Mn bind to each HDL particle (when
calculated for a particle of 360 kDa of which 45% is protein).
Corrole distribution in plasma. The distribution of corroles in plasma was evaluated
by adding either 20 or 40 µM of 1-Mn or 1-Fe to 4 mL plasma from healthy
volunteers. After 30 minutes of equilibration, the mixtures were treated for 48 hours
by ultracentrifugation in a KBr density gradient as previously described[3]. Fractions
of VLDL, LDL, HDL and LPDS were collected, and the electronic spectrum of each
fraction was recorded (Figure S2). Plasma fractions without corroles were used as
reference. The amount of cholesterol in the fractions was determined by the
CHOL/PAP kit (Roche/Hitachi) and normalized according to fraction volume.
Oxidation of LDL by peroxynitrite. LDL (100 mg protein/L) in PBS was incubated
for 30 min at room temperature without any additive or with 1-Mn or 1-Fe (5 µM).
LDL oxidation was induced by addition of SIN-1 (250 µM) and incubation for 4 hour
at 37 ºC under air in a PowerWavex Microplate Scanning Spectrophotometer (Bio-Tek
Instruments Inc.) equipped with a KC4 software. LDL oxidation was continuously
monitored by measuring the formation of conjugated dienes, as indicated by the
increase in absorbance at 234 nm[5].
Oxidation of LDL/HDL by copper sulphate. LDL/HDL (100 mg protein/L)
solutions in PBS were incubated for 30 min at room temperature without any additive
or with 1-Mn, 1-Fe or 1-Ga at various concentrations (0.5, 2.5 and 5 µM). Oxidation
was initiated by addition of a freshly prepared CuSO4 solution (5 µM) and incubation
(at 37 ºC under air in a shaking water bath) was continued for 2 h (LDL) or 5 h
(HDL). Lipoprotein oxidation was determined by measuring the amount of TBARS[5].
Kinetic measurements were performed in a similar manner, both without any additive
and with 2.5 µM 1-Mn or 1-Fe. Conjugated dienes formation was continuous
monitored for 165 min while TBARS and lipid peroxides were measured as
previously described[5] at the times shown in the graphs.
Corrole-mediated efflux from J-774 macrophages. Murine J-774 cells (1x106/mL)
were plated in 24-well plates for 24 hours, then washed and incubated for 1 hour in
serum-free DMEM that contained 3H-cholesterol (2 µCi/mL) and BSA (0.2%). Cells
were washed to remove unincorporated label and then incubated in 1 mL of DMEM
without any additive or with 10, 25 or 50 µM of 1-Mn, 1-Fe or 1-Ga. After a 4-hour
incubation at 37 °C to permit efflux of 3H-cholesterol from the cells into the medium,
500 µL of the medium was collected. The cells were washed with PBS, 1 mL of 0.1 N
NaOH was added to the cells and 500 µL was collected the next day. Medium and
cellular 3H-cholesterol were determined by liquid scintillation counting (LSC). The
percentage of cholesterol efflux was calculated as the ratio of total counts per minute
in the medium divided by the total counts per minute in the medium and in the cells.
Corrole-mediated cholesterol efflux was calculated after subtraction of the nonspecific efflux obtained in cells incubated in the absence of corroles.
Experiments with E0 (apolipoprotein E deficient) mice. At an age of 12 weeks, 24
male E0 mice were divided randomly to 4 groups of 6 mice each. The groups differed
only in the type of drinking water: no additive, and water containing 0.04 mM of
either 1-Mn, 1-Fe or 1-Ga. Fluid consumption by the groups receiving 1-Mn and 1Ga was ~ 5 mL/mouse/day, which equals to 0.2 mg per mouse per day. The group
receiving 1-Fe was found to drink somewhat larger amounts (~6 mL/mouse/day).
After 10 weeks the mice were sacrificed and blood samples, heart with attached aorta
and mouse peritoneal macrophages (MPM) were collected from all mice.
Serum lipids. Serum samples from all mice in the same group were mixed and
submitted for analysis at the “Chemistry laboratory” at the Rambam Medical Center,
Haifa.
Macrophage paraoxonase 2 (PON2) activity. Mouse peritoneal macrophages
(MPM) were harvested 4 days after intra-peritoneal injection of 3 mL thioglycolate
(40 g/L). The cells were washed with PBS, diluted to 106 cells/mL in DMEM
supplemented with fetal calf serum, plated and incubated at 5% CO2 and 37 ºC.
Dihydrocoumarin was utilized as substrate for measuring PON2 lactonase activity as
previously described
[6]
. The cells (2·106) were washed and incubated with 1 mL of 1
mM dihydrocoumarin in Tris buffer. After 10 min incubation at room temperature, the
absorbance at 270 nm was measured. The self-hydrolysis of dihydrocoumarin was
measured (and subtracted) under the same conditions in a cell-free system for
calculating the cell-mediated hydrolysis of dihydrocoumarin.
Histopathology development of aortic atherosclerosis lesions. Heart and entire
aorta were rapidly dissected out from each mouse and immersion-fixed in 3%
glutaraldehyde in 0.1 M sodium cacodylate buffer with 0.01% calcium chloride, pH
7.4, at room temperature. The first 4 mm of the aortic arch was stained with osmium
tetroxide, which colors all the lipid components a dark brown-black color thus
enabling delineation of the lesion with greater accuracy. The blocks were embedded
in epon resin and thin transverse sections were cut to allow greater resolution of the
lesion details. The area covered by the lesion was determined by image analysis [7].
Oxidation of DMSO by copper sulfate.
Concentrations (M) of formaldehyde that were produced
Reducing agent
No additive
1-Mn
1-Fe
Sodium
ascorbate
5·10-5
1.6·10-5
0
Glutathione
1.6·10-5
0.6·10-5
0
Figure S1. The electronic spectra of the fractions collected from the density
gradient treated serum (see Figure 1), shown after abstraction of the
contributions from non-corrole containing fractions. The percentage of HDLbound corrole was estimated by ignoring possible variations in the extinction
coefficients of the molecules under the different environments.
1.8
absorbance @ 234 nm
1.6
1.4
1.2
1
0.8
0
60
120
180 240
300 360
time [min]
Figure S2. The effect of corrole (2.5 µM) on SIN-1 induced (200 µM)
oxidation of LDL (100 mg protein/L) as measured by conjugated dienes
formation (absorbance at 234 nm):
without corrole;
with 1-Mn; and
with 1-Fe.
[1]
[2]
[3]
[4]
[5]
[6]
[7]
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