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Modulation of Erythrocyte Potassium Chloride Cotransport, Potassium
Content, and Density by Dietary Magnesium Intake in
Transgenic SAD Mouse
By Lucia
De Franceschi, Yves Beuzard, Helene Jouault, and Carlo Brugnara
Prevention of erythrocyte dehydration is a potential therapeutic strategy for sickle cell disease. Increasing erythrocyte
magnesium (Mg) could inhibit sickle cell dehydration by increasing chloride (CI) and water content and by inhibiting
potassium chloride (K-CI) cotransport. In transgenic SAD 1
and (control) C57BL/6 normal mice, we investigated the effect of 2 weeks of diet with either low M g (6 f 2 mg/kg
body weight/d) or high M g (1.000 f 20 mg/kg body weight1
d), in comparison with a diet of standard M g (400 f 20 m g l
kg body weight/d). The high-Mg diet increased SAD 1 erythrocyte M g and K contents and reduced K-CI cotransport activity, mean corpuscular hemoglobin concentration (MCHC),
cell density, and reticulocyte count. SAD 1 mice treated with
low-Mg diet showed a significant reduction in erythrocyte
M g and K contents and increases in K-CI cotransport, MCHC,
cell density, and reticulocyte counts. In SAD 1 mice, hematocrit (Hct) and hemoglobin (Hbl decreased significantly with
low M g diet and increased significantly with high-Mg diet.
The C57BLJ6 controls showed significant changes only in
erythrocyte M g and K content, and K-CI cotransport activities, similar to those observed in SAD 1 mice. Thus, in the
SAD 1 mouse, changes in dietary M g modulate K-CI cotransport, modify erythrocyte dehydration, and ultimately affect
Hb levels.
0 1996 by The American Society of Hematology.
T
tors of this pathway that can be used to prevent cell dehydration in vivo. However, K-Cl cotransport is exquisitely sensitive to cell magnesium (Mg) concentration, and a modest
increase in cell Mg induces marked inhibition of K-C1 cotransport.''
The Mg content of the red blood cell is an essential modulator of red blood cell volume, volume regulatory mechanisms, and membrane functions. Erythrocyte Mg content
modulates the activities of the Na-K pump, the Na-K-Cl
cotransport, the K-C1 cotransport, the Ca and K channels,
and affects cell membrane structure and function.I4 The cellular Mg content has a direct effect on erythrocyte volume
and water content. When cell Mg is increased, C1 moves
into the cell to compensate the positively charged Mg ions
with osmotically obligated water influx and consequent cell
swelling. There have been reports of abnormally low cell
Mg content in sickle erythrocytes, especially in the dense
fractions containing irreversibly sickle cells (ISC).''."
Based on the available evidence, increasing erythrocyte
Mg content of sickle cells would produce cell swelling by
two different mechanisms: first, osmotic increase of cell water content and second, increased K content via inhibition
of K-CI cotransport." To test this hypothesis, we investigated the effects of a 2-week treatment with diets containing
different amounts of Mg in a transgenic mouse model for
sickle cell disease, (SAD
and in control C57BL/6
mice. Our results suggest that in SAD 1 and C57BL/6 mice
erythrocyte Mg content varies according to the Mg dietary
intake. Furthermore, changes in intracellular Mg content
modulate K-Cl cotransport activity, and modify red blood
cell K content and density. Hb levels in SAD mice change
according to dietary Mg intake. Thus, dietary Mg supplements may be a potential therapeutic strategy to prevent
dehydration of SS cells in vivo.
HE POLYMERIZATION tendency of hemoglobin (Hb)
S-containing erythrocytes (SS erythrocytes) is exponentially related to the cellular concentration of Hb S to its
20th to 40th power.' The presence of dense cells containing
polymerized Hb S has been linked to the clinical severity of
various sickle syndromes.' Thus, the potassium (K) lossinduced dehydration observed in SS erythrocytes is an important contributor to the pathogenesis of the disease. In
vitro data suggest that inhibition of cell dehydration could
be achieved via blockade of the two major erythrocyte K
efflux pathways: the K-chloride (C1) cotransport system".'
and the Ca-activated K
The Ca-activated K channel (Gardos pathway7) is activated by an increase in intracellular free calcium ion. The opening of the channel promotes
K and CI loss and cell dehydration. It is likely that the
physical distortion of the red blood cell membrane induced
by Hb S polymerization leads to a transient increase in free
intracellular Ca, which activates the Gardos pathway, with
loss of cell K and dehydration. The clinical utility of
blockading this channel by oral administration of clotrimazole is currently being investigated.x-"' The K-CI cotransport
system promotes loss of K and C1 with consequent erythrocyte dehydration when the cells are exposed to pH values
lower than 7.40.1'.'2There exist no pharmacological inhibi-
From the Department of Internal Medicine, University o j Verona,
Verona, Italy: Luboratoroire d'Hemutologie and INSERM Unit 91,
Hospital H Mondor, Creteil, France; and the Department of Laboratory Medicine, The Children's Hospital, Harvard Medical School,
Boston, MA.
Submitted January 30, 1996; accepted May 21, 1996.
Supported by National Institutes of Health grants from the Heart.
Lung and Blood Institute (P60-HLl5157) and from the Diabetes,
and Digestive, and Kidney Diseases Institute (ROI -DK50422).
Address reprint requests to Carlo Brugnara, MD, Department of
Laboratory Medicine, The Children's Hospital, 300 Longwood Ave,
Bader 760, Boston, MA 02115.
The publication costs of this article were defrayed in part by page
charge payment. This article must therejbre he hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8807-0009$3.00/0
2738
MATERIALS AND METHODS
Drug3 and chemicals. NaCI, KCI, ouabain, bumetanide, Tris
(hydroxymethyl), Tris (aminomethane), 3(N-morpholino) propanesulfonic acid (MOPS), choline chloride, MgC12, and Acationox
were purchased from Sigma Chemical Co, (St Louis, MO). MgCl,,
dimethyl-sulfoxide (DMSO), sulfamic acid (SFa), n-butyl phthalate
and all other chemicals were purchased from Fisher Scientific Co
(Fair Lawn, NJ). Microhematocrit tubes were purchased from DrumBlood, Vol 88,No 7 (October I ) , 1996:pp 2738-2744
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2739
MAGNESIUM THERAPY IN SAD MOUSE
Table 1. Effects of a Two-Week Course With Different Mg Dietary Intakes on Serum
and Intracellular M g Content in C57BL/6 and Transgenic SAD 1 Mice
SADl
C57BU6
Low-Mg Diet
Low-Mg Diet
High-Mg Diet
Erythrocyte
(mmollkg Hb)
(mmol1L)
Erythrocyte
(mmollkg Hb)
Erythrocyte
(mmol1L)
Erythrocyte
b-r"l/kg Hb)
Serum
(mmol1L)
(mmolIL)
(mmollkg Hbl
2.7 c 0.2 ( 6 )
1.3 c 0.1 (6)*
9.2 f 0.4 ( 6 )
7.2 f 0.1 ( 6 ) t
2.8 t 0.1 (6)
4.7 -c 0.2 ( 6 ) t
8.9 f 0.3 ( 6 )
9.6 t 0.2 (6)t
2.2 f 0.2 (6)
1.2 t 0.2 ( 4 ) t
7.5 f 0.1 ( 6 )
6.0 f 0.5 ( 4 ) t
2.1 t 0.3 (6)
5.6 f 0.1 (4)t
7.3 t 0.2 ( 6 )
9.6 t 0.9 (4)t
Serum
Serum
Serum
High-Mg Diet
Time (d)
0
14
Data are presented as means f S D ( n of determinations).
* P i .05 compared with baseline values.
t P < .005 compared with baseline values.
mond Scientific Co. (Bromall, PA). All solutions were prepared
using double-distilled water.
Animals and experimental design. Eighteen transgenic Hbbs'"@'/
Hbbsl"g'eSADl (SAD 1) mice were used for the experiment with 18
C57BL/6 normal mice composing the control group. All of the mice
were obtained from breeding performed in the animal facility of
INSERM at Henri Mondor Hospital (Creteil, France). The selected
mice, between 4 and 6 months of age, were both female (weight 25
to 28 g) and male (weight 28 to 30 g).
The low-Mg group received a diet that included 6 ? mg Mg/g
body weightld. This was achieved by using a mouse feed with an Mg
content of 30 2 10 mglg, provided by Uar, Villemoisson, Epinay-s/
Orge, France.
The standard Mg group received a diet that included 400 2 20
mg of Mglg body weight/d. This was achieved with a regular mouse
feed with an Mg content of 1,900 ? 100 mg/g.
The high-Mg group received a diet that included 1,ooO 5 20 mg
of Mg/g body weight/d. This was achieved by supplementing the
Mg contained in the regular mouse feed with an additional 600 mg
Mg/g body weight/d. This Mg supplement consisted of magnesium
hydroxide dissolved in water and was administered by gavage. Normal daily Mg intake for humans is 418 ? 120 mg in males and 343
2 94 mg in females."
Measurements of the six groups were performed at baseline and
after 14 days of treatment. No changes in body weight were observed
during the treatment. At specific times, 200 pL of blood were drawn
from each animal and used for assays of K-CI cotransport activity,
erythrocyte phthalate density distribution curves. erythrocyte cation
content, and other hematological parameters.
Two SAD 1 mice in the high-Mg diet group died during the first
week of treatment because of traumatic complications of the gavage
procedure. No cause of death was determined for the two SAD mice
of the low-Mg diet group that also died during the first week of the
study. No C57BL/6 controls died during the study.
Hematological data and cation contents. Blood was collected
from ether-anesthetized mice by retro-orbital venipuncture into heparinized microhematocrit tubes. Hb concentration was determined by
spectroscopic measurement of the cyanmet derivative. Hematocrit
(Hct) was determined by centrifugation in micro-Hct centrifuge.
Mean corpuscular hemoglobin concentration (MCHC) was calculated from the measured Hb and Hct values. Reticulocytes were
counted on a Coulter EPICS Profile I1 (Coulter Electronics, Hialeah,
FL) after thiazole orange staining: 2.5 pL of whole blood was incubated with 0.1 mg of thiazole orange in 1 mL, filtered phosphatebuffered saline (PBS) buffer for 30 minutes. The fluorescence of
50,000 erythrocytes was collected with log amplification."
Density distribution curves were obtained according to Danon and
M a r i k o v ~ k yusing
, ~ ~ phthalate esters in microhematocrit tubes, after
washing the cells three times with PBS solution (300 mOsm) at
25°C in 2-pL tubes. The remaining cells were washed four additional
times with choline washing solution (170 mmol/L choline, 1 mmoY
L MgClz, Tris-MOPS pH 7.4 at 4"C, 330 mOsm) for measurements
of internal Na and K contents by atomic absorption spectrometry.
Measurements of K-C1 cotransport activity in mouse red blood
cells. Activity of K-CI cotransport in fresh mouse erythrocytes was
measured as the chloride-dependent K efflux and volume-dependent
K e f f l u ~ . Net
~ ~ ~K~efflux
'
from fresh cells was measured in isotonic
(340 mOsm) and hypotonic (260 mOsm) Na media. A level of 340
mOsm is considered isotonic for mouse red blood cells.24Chloridedependent K fluxes were calculated as the differences between K
efflux values in chloride and in sulfamate hypotonic media. All flux
media contained (in mmoVL) 1 MgC12, 10 glucose, 1 ouabain, 0.01
bumetanide, and 10 Tris-MOPS (pH 7.40 at 37°C). Efflux was calculated from the measured supernatant K concentrations at 5 and 25
minutes.
Statistical analysis. All values are means 2 standard deviation
(SD). For each group of mice, comparisons of the separate variables
between baseline state and after 14 days of treatment were performed
using two-tailed Student's t-test. Comparison of more than two
groups was performed by one-way analysis of variance (ANOVA)
with Tukey's test for posr hoc comparison of the meansz6 Correlations were assessed by calculation of Pearson's correlation coefficient.
RESULTS
Esfects of difSerent Mg dietary intakes on serum and erythrocyte Mg levels in normal and SAD I mice. At baseline,
when all the mouse groups were under standard Mg diet,
the SAD 1 mouse red blood cells showed lower intracellular
Mg content and lower serum Mg levels compared with the
control C57BL/6 strain (Table 1).
After 14 days of treatment with low-Mg diet, there was a
significant decrease in serum and red blood cell Mg content
compared with standard diet in both SAD 1 and the C57BU
6 control mice ( P < .05; Table 1). Although a low-Mg diet
abolished differences in serum Mg between the two strains,
a significantly lower ( P < .05) erythrocyte Mg content persisted in the SAD 1 mice compared with the corresponding
controls (Table 1).
In SAD 1 and C57BL/6 control mice, a high-Mg diet
resulted in a significant increase in both serum and erythrocyte Mg levels compared with a standard diet ( P < .05;
Table 1). With a high-Mg diet, serum and red blood cell Mg
of SAD 1 mice achieved values similar to those observed in
C57BW6 control. Thus, a high-Mg diet abolishes the lower
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2740
serum Mg and the reduction in erythrocyte Mg content observed in SAD l mice.
Effects of different Mg dietary intakes on hematological
parameters of C57BU6 and SAD 1 mice. After 14 days of
treatment with a low-Mg diet, transgenic SAD 1 mice
showed a significant reduction in Hct and Hb and an increase
in percent reticulocytes, MCHC, and average red blood cell
density (D50)compared with mouse groups treated with standard ( P < .05) and high-Mg2+diet ( P < .002; Tables 2 and
3). In C57BL/6 control mice exposed to low-Mg" diet, an
increase in the percent reticulocytes and in the average red
blood cell density, measured as D50 withthe phthalate
method, were observed, with no significant changes in either
Hb, Hct, or MCHC (Tables 2 and 3).
In SAD 1 mice, a high-Mg diet yielded a significant increase in Hct and Hb, and a significant decrease in percent
reticulocyte, MCHC, and Dso compared with a standard Mg
diet (Tables 2 and 3). In the C57BW6 control group treated
withhigh-Mg diet, only a significant increase in Hb was
noted, withno changes in reticulocyte counts, MCHC, or
D5,,
(Tables 2 and 3).
Marked changes in red blood cell morphology were observed in SAD 1 mice when the dietary Mg intake was
varied. C57BL/6 control mice fed with the low-Mg diet exhibited abnormalities in 5% to 15% of erythrocytes, with
small size, irregular shape, or loss of biconcavity (data not
shown). SAD 1 mice exposed tothe low-Mg diet had a
larger proportion of these abnormal cells (20% to 40%) and,
in addition, elongated cells resembling human irreversibly
sickled cells (Fig 1). The high-Mg diet did not change the
morphology of either control or SAD 1 erythrocytes.
Effects of drfferent Mg dietary intakes on red blood cell
cation content and K-Cl cotransport activity in normal and
SAD I mice. At baseline, SAD 1 red blood cells showed
a lower K content and increased K-Cl cotransport activity
compared with C57BL/6 erythrocyte (Table 4 and Fig 2).
This is consistent with previous reports on the SAD l mouse
model.',"
Exposure for 14 days to a low-Mg diet determined a significant increase of K-Cl cotransport activity in both SAD
1 and C57BL/6 control mice compared with standard ( P <
.05) and high-Mg" diet ( P < .005).Conversely, SAD 1 and
C57BL/6 control mice treated with a high-Mg diet demonstrated a decreased K-Cl cotransport compared with animals
fed with the standard diet (Fig 2).
These changes in K-Cl cotransport activity were associated
with changes in the red blood cell K content. A low-Mg diet
induced a significant depletion in erythrocyte
K content in both
SAD 1 and C57BU6 control mice when compared with either
standard diet ( P < .02) or high-Mg diet ( P < .005; Table 4).
Erythrocyte K content increased significantly whenSAD 1 and
C57BU6 control mice were treated with a high-Mg diet ( P <
.05; Table 4). Onlyin SAD 1 micewastheincreasein
K
content of a magnitude to induce measurable changes in MCHC
and D5"(see Tables 2 and 3).
No significant changes of red blood cell Na content of
C57BL/6 mice were observed in this study (Table 4). In
SAD 1 mice, a low-Mg diet resulted in a slightly increased
erythrocyte Na' content compared with baseline conditions
( P < .05; Table 4).
DE FRANCESCHI ET AL
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2741
MAGNESIUM THERAPY IN SAD MOUSE
Table 3. Effects of a Two-Week Course With Different Mg Dietary Intakes on MCHC and Dm in C57BL16 and in Transgenic SAD 1 Mice
SAD 1
C57BM
Low-Mg Diet
Time (d)
0
14
High-Mg Diet
Low-Mg Diet
High-Mg Diet
MCHC (g/dL)
D50
MCHC (g/dL)
Dm
MCHC (g/dL)
050
MCHC (g/dL)
050
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31.5 c 0.8 (6)
1.090 2 0.001 (6)
1.094 c 0.001 (6).
31.2 c 0.5 (6)
29.8 c 1.6 (6)
1.090 z 0.001 (6)
1.088 c 0.002 (6)
35.7 2 0.2 (6)
37.1 c 0.7 (4)t
1.106 c 0.002 (6)
1.114 2 0.001 (4).
36.1 c 0.1 (6)
32.7 2 0.4 (4).
1.106 c 0.002 (6)
1.101 2 0.001 (4).
Data are presented as mean 2 SD ( n of determinations).
P < .005 compared with baseline.
t P < .05 compared with baseline.
DISCUSSION
This report demonstrates that changes in dietary Mg intake
modulate serum and red blood cell Mg levels, affect red
blood cell K-CI cotransport activity, and K content in both
SAD 1 and C57BU6 mice.
When the daily Mg intake was reduced from approximately 400 2 IO mg M a g body weight/d to approximately
6 2 2 mg Mgkg body weight/d, significant reductions in
serum and red blood cell Mg levels were observed in both
SAD 1 and C57BU6 mice. Significant changes in ion composition were observed with increased Na contents, MCHC,
red blood cell density, K-CI cotransport activity, and with
a reduced erythrocyte K content (Tables 2 through 4). As
described in Mg-deficient rabbits, Mg deficiency may cause
a decreased enzymatic activity of the Na/K ATPase and thus
lead to increased cell Na and decreased cell K contents.”
We did not measure Na-K ATPase activity in this study.
However, because the reduction in cell K greatly exceeds
the gain in cell Na, the most likely determinant of the cell K
depletion induced by dietary Mg deficiency is the increased
activity of K-CI cotransport and not a change in Na-K pump
activity (Fig 2).
In SAD 1 mice, a low Mg diet determined a significant
decrease in Hct and Hb levels compared with the standard
diet. This was associated with changes in red blood cell
morphology such as loss of biconcavity and elongated shapes
(Fig 1). These morphological abnormalities, the increased
reticulocyte count, and the decreased Hct and Hb suggest a
reduction in erythrocyte survival time in circulation and indicate that intracellular Mg may play an important role in red
Fig 1. Effect of dietary Mg on
erythrocyte morphology: Red
blood cell morphology in SAD 1
mice after 14 days of high-Mg
diet (A) and low-Mg diet (B).
blood cell life span. A similar role has been shown for Mg
in rat erythrocytes.**Dietary Mg deficiency in rats has been
shown to produce increased osmotic fragility, increased
membrane fluidity, and decreased cell K c~ntent.2~
A diet enriched in Mg resulted in significant increases in
serum and red blood cell Mg levels for both SAD 1 and
C57BU6 mice (Table I). In SAD 1 mice, these changes were
associated with a significant reduction in K-CI cotransport
activity (Fig 2). decreased MCHC, decreased cell density
(Table 3), and increased erythrocyte K content (Table 4).
There were also significant increases in Hct and Hb and
reductions in reticulocyte counts in the SAD 1 mice treated
with high-Mg diet, possibly indicating reduced hemolysis
and increased erythrocyte survival (Table 2).
The changes in red blood cell composition induced by
manipulations of Mg intake are relevant to the pathophysiology of sickle cell disease because of the high-order exponential dependence of the delay time for Hb S polymerization
on the concentration of Hb S.’ Cell dehydration will result
in an increase in intracellular Hb S concentration with a
disproportionate reduction in the delay time, acceleration of
Hb S polymerization, increased cell sickling, and vasoocclusion. Prevention of cell dehydration is a possible therapeutic
strategy for decreasing Hb S polymerization and cell sickling
in patients with SS disease. Theoretically, this can be
achieved by either promotion of osmotic swelling? or by
pharmacological prevention of the loss of cell K, which is
the main determinant of SS cell dehydration. Clotrimazole,
an imidazole antifungal agent and specific blocker of the
Ca-activated (Gardos) K channel, is currently undergoing
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
DE FRANCESCHI ET AL
2742
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(bottom) mice at baseline (4)and after 14 days (m) at three different
dietary M g intakes.
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clinical studies in patients with S S disease.'" Because it is
theoretically possible that blockade of only one of the two
pathways involved in sickle cell dehydration could lead to
a compensatory response by the other pathway, combined
pharmacological inhibition of both pathways is an interesting
possibility. There are no specific pharmacological inhibitors
of the K-Cl cotransport. However, it had been demonstrated
previously that increased concentrations of cell Mg inhibit
the activity of the WC1 cotransporter in SS cells and thereby
increase cell volume in vitro." This effect is present in the
least dense fraction of normal AA cells, which havean intrinsically high rate of K-Cl cotransport.3' The increased free
Mglevel secondary to hemoglobin binding to its organic
phosphate chelators is responsible for the inhibition by deoxygenation of volume sensitive WC1 cotransport in both S S
and AA cells?' Lower levels of red blood cell Mgin patients
with S S disease compared with control individuals have been
reported. If'
There may be a component of genetic control for erythrocyte magnesium in normalmales since lower erythrocyte
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MAGNESIUM THERAPY IN SAD MOUSE
Mg contents have been observed in HLA-B35 carriers.33It
is intriguing to postulate a connection to the observation that
certain sickle cell complications are more frequent among
patients with HLA-B35.34
A few studies have shown that oral Mg supplements can
successfully increase erythrocyte Mg level^,'^.^^ whereas
other studies have not.37There have been some uncontrolled
reports of a beneficial effect of Mg in patients with S S disHowever, a 7-day course of Mg supplements did
not show any change in red blood cell survival in three
patients with SS disease!’
Recently, Franco et a14’ have studied the activity of K/
C1 cotransport in transfemn receptor positive (TfR+) dense
reticulocytes. They showed that the red blood cells, which
become dense quickly in vivo, have more K-Cl cotransport
activity than those which remain light in vivo, indicating KC1 cotransport as the primary mechanism for dehydration of
young sickle cells. Inhibition of K-C1 cotransport in young
dense sickle cells by Mg may prevent this fast track red
blood cell dehydration and complement the inhibition of the
dehydration of mature red blood cells obtained by blocking
the Gardos channel with clotrimazole.”
In conclusion, these results show that Mg dietary intake
affects serum and red blood cell Mg content in SAD 1 and
C57BL/6 mice. An Mg-deficient diet leads to worsening
anemia, reticulocytosis, and increased dehydration of SAD
1 mouse red blood cells, most likely mediated by increased
K-Cl cotransport. A high-Mg diet decreases K-C1 cotransport
activity, red blood cell dehydration, and K loss of transgenic
SAD 1 mouse red blood cells and increases Hb levels, suggesting a possible amelioration of the disease.
Oral Mg supplementation is associated with rare, mild
gastrointestinal side effects (mostly cramps) and diarrhea at
high dosages. These data in mice provide the rationale for
studying the intake of Mg in patients with sickle cell anemia
and the effects of dietary Mg supplements in sickle cell
disease.
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1996 88: 2738-2744
Modulation of erythrocyte potassium chloride cotransport, potassium
content, and density by dietary magnesium intake in transgenic SAD
mouse
L De Franceschi, Y Beuzard, H Jouault and C Brugnara
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