From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
Acid
pH
Induces
Formation
By Carlo
When
erythrocytes
globin
S (SS)
K. CI.
and
55
for
70)
at
O
a volume
cells
to 12
constant
cell
NE
OF
SS
and
is absent
(MCHC;
content
40 to 50 g/dL)
probably
contribute
most
of the
of the
understood,
tion.
incu-
<7.40
from
in
(7.3
the
This
cell (55)
disease.2’3
cells
has been
Deoxygenation
may
a 1989
arated
or
physiologic
formation
of dense
cells
through
loss of K through
the Ca-activated
K channel510
or
as a consequence
of the increased
Na entry
induced
by
deoxygenation,
which
leads to activation
of the Na/K
pump
and
results
in
stoichiometry
investigators
cell
dehydration
of this
transport
have
changes
namely
reported
due
to
sy5tem.”3
that
the
in cation
permeability
the
increased
K effiux
the
3Na,/2K1
However,
deoxygenation-induced
of the RBC
membrane,
and
Na
influx,’4”5
are
cation
and
oxidative
damage
as possible
RBC
membrane
mechanisms
of K loss and
We recently
described
erythrocytes
or shear
stress
55 cell dehydration)7”8
in carbon
monoxide-treated
the presence
of a KC1 cotransport
55
system
RBCs
of patients
homozygous
for hemoglobin
C (CC),2’
in
the least dense fraction
of normal
(AA)
cells,22’23 and in LK
RBCs
this
K transport
densest
HK
sheep
charged
cotransport
densest
which
have
confirmed
movement
in the
of normal
determinant.
positively
the KC1
reticulocytes.24
pathway
fraction
important
the
and
cells
We
least
suggests
have
hemoglobins
since this
also
The
but
that
cell
fractions
of CC
life span.25
cells
and
Work
cells
is activation
system
by
severe
exercise
denser
acid
pH.
and
every
tion,
time RBCs
the increased
Blood,
Vol
74,
No
Indeed,
systemic
release
CO2
1 (July),
with
plasma
or localized
pp 487-495
the
5S2
fraction.27
552
fraction
added.
We
formation
and
cells
of
<7.40.
The
dense
the
because
separated
effect
shrinkage;
confoundsickling,
with
55
formed
unsep-
to mimic
inhibitors.
cell
dense
the
either
saturated
cells
Their
of cells
we examined
in 55
KC1
cell
investigated
55 cells,
S polymerization
et al
of this
induces
in acid-induced
cell
AA cells. To avoid
cells
we
carbon
can
be
deoxygenation,
55 cells
in
by exposure
to
of loss of K, Cl, and
according
to density,
formation
of dense cells is maximal
in the least
and is absent
in the densest
fraction.
These
suggest
that
system
induced
by acidification
SS cells
K
in the
loss
through
the
KC1
contributes
absence
MATERIALS
cotransport
to formation
of
of deoxygenation.
AND
METHODS
Preparation
of erythrocytes.
After we obtained
informed
consent, we collected
blood in heparinized
Vacutainer
tubes (Becton
Dickinson,
Rutherford,
NJ) from donors homozygous
for hemoglobin S (55 cells) and normal controls
(AA cells). Leukocytes
were
removed by cotton filtration.
The blood was centrifuged
in a Sorvall
From
Hospital;
in AC
cells,
that
the
is markedly
K
the
during
Moreover,
the
Supported
the
by
National
The
charge
reprint
St.
No.
Lung
C1-607.
Boston.
accepted
2-P60-HL15157
Brugnara.
MA
Women’s
Physiol-
from
Department
Medical
of
School.
25
02115.
ofthis
article
payment.
This
article
must
with
MD.
Harvard
costs
I 989
8. 1989.
and
to Carlo
Boston,
and
Molecular
March
Institute.
Physiology.
in accordance
and
Blood
publication
indicate
Brigham
HL-36076
and
requests
Molecular
“advertisement”
©
Grants
Pathology.
of Cellular
School,
6. /988;
Heart,
and
Shattuck
of
Department
Medical
October
Address
55
Department
and
ogy. Harvard
Submitted
et al has
oxygen
in the peripheral
circulaconcentration
lowers
plasma
and
1989:
Canessa
density.
In the attempt
we did not use transport
results
of
in
decreases
of the
on
high MCHC,
55 cells are
acid-induced
dense
fraction
for
by which
acidosis.
to Hb
pH
in
is an
of the KC1 cotransport
pH
swelling.
of KC1 cotransport,
that
acid pH have
water.
When
Cellular
one of the mechanisms
the role
by
due
the activity
inactivated
by Canessa
stimulated
by NEM.26’27
We hypothesized
that
become
of
in the
youth
separated
conditions,
all experiments
a role
is present
and extended
our findings,
showing
of 55 cells is chloride
dependent
and
may
not
postulated
in determining
pathway
is not
a normal
presence
dense
cells
ouabain
performed
media
co-
cotransport
that activation
by acid
pH
nystatin-treated
ing factors
dense
stimu-
lated by acid pH and cell swelling
and inhibited
by internal
Mg)9’2#{176}
The activation
ofthis
system
by cell swelling
induces
K, Cl, and water
loss. A similar
system
is also present
in
sheep
of the
and
monoxide.
We showed
no KCI
can form
KCI
accomplished
in vitro,
in the absence
of
merely
by incubating
carbon
monoxide-treated
other
balanced
and do not produce
changes
in the total
water
content.’6
Other
studies
have
suggested
of the
cell
some evidence
in 55 cells
with
of fresh
To prove
co-
in the
Inc.
of external
K concentration
we also used density-separated
deoxygena-
have
cells
the
dense
KCI
density
which
dense
when
performed
in air
maintain
in cell
that
corresponding
were
which
with
with
in 27%
behavior
least
by acidification.
shrinkage
incubated
according
in the
considerable
erythrocyte,
suggest
& Stratton.
reported
system
comprise
has
erythrocytes
previously
cotransport
cells
separated
no change
of AA
is activated
are
shrinkage
which
data
by Grune
pH,
cells
cell
produces
55
cellular
cation
AA
cells.
These
dense
(ISC),
AA
fractions
transport.
system
When
pH induces
but
experiments
associated
induce
of
Erythrocytes
C. Tosteson
acid
fraction
the
shape
even
when
fully
oxygenated.4
The
cells formation
in 55 disease
is not yet well
but usually
fraction.
to density.
water
and potassium
to the vasoocclusive
These
sickled
densest
oxygenated
disease
and
Daniel
and
from
volume
in Sickle
Ha, and
densest
to
top
cells
Cells
transport.
with
markedly
increased
hemoglobin
concentration
and reduced
significantly
irreversibly
their
deformed
process
of dense
cells
ofsickle
of erythrocytes.’
RBCs
and
mean
corpuscular
manifestations
when
pH
in cell
content
density
KCI
(DPG).
in fresh
heterogeneity
lose
monoxide-
denser
with
HALLMARKS
marked
carbon
Van
hemo-
they
pH-dependent
that
in media
fractions.
THE
is the
for
2.3-diphosphoglycerate
is maximal
density
and
progressively
hours
Thuong
to pH <7.40.
report
become
eight
phenomenon
middle
through
We
Brugnara,
homozygous
or exposed
system.
treated
patients
swollen
water
cotransport
bated
from
are
of Dense
were
defrayed
therefore
18 U.S.C.
be
section
in part
hereby
by page
marked
1 734 solely
to
this fact.
by Grune
& Stratton,
Inc.
0006-4971/89/7401-0043$3.00/0
487
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
488
refrigerated
centrifuge
(RB SB, Du Pont, Sorvall Biomedical,
Newtown, CT) at 5#{176}C
for ten minutes at 3,000 g; cells were washed five
times with a washing
solution
containing
152 mmol/L
choline
chloride,
1 mmol/L
MgC12,
10 mmol/L
Tris-3(N-morpholino)
propanesulfonic
acid (MOPS),
pH 7.4 at 4#{176}C.
The erythrocytes
were then suspended
at 10% hematocrit
(Hct) in choline washing
solution at 4#{176}C
and were equilibrated
for 20 minutes with CO. which
was bubbled
in a flask containing
choline washing
solution at 4#{176}C.
An aliquot of cells was then suspended
in an approximately
equal
volume
of choline
washing
solution;
from this cell suspension,
determinations
of Hct, cell Na (1:50 dilution
in 0.02% Acationox,
American
Scientific
Products,
McGaw
Park, IL), cell K (1:500
dilution),
hemoglobin
(Hb, optical density at 540 nm in Drabkin’s
solution),
MCHC,
and phthalate
density profile
were performed.
Since phthalate
densities
are markedly
temperature
dependent,
the
RBCs were equilibrated
at room temperature
before centrifugation.
Centrifugation
lasted six to seven minutes,
and 15 minutes elapsed
between each centrifugation
to allow cooling down of the microcentrifuge head. Median
density (D) and middle 60% density range
(R60) of phthalate
density
profiles were determined
according
to
Rodgers et al. The erythrocyte
Na and K contents were determined
in a Perkin-Elmer
atomic
absorption
spectrophotometer
(model
5000, Perkin-Elmer,
Norwalk,
CT) using standards
in doubledistilled water.
The nystatin
loading
procedure
was used to modify the RBC
cation content as detailed
in previous
publications.21’22
Recovery
of
normal permeability
to cations after nystatin treatment
was assessed
by measuring
the rate constant
for K efflux from fresh cells and
nystatin-loaded
cells with composition
and MCHC
similar to fresh
cells. K efflux was measured
at 1% Hct as specified
in previous
publications.2U2
Flux media contained
140 mmol/L
NaCI or 100
mmol/L
NaCI and 0 to 100 mmol/L
choline chloride,
I mmol/L
MgCI2,
10 mmol/L
glucose,
0.1 mmol/L
ouabain,
0.01 mmol/L
bumetanide,
and 10 mmol/L
Tris-2(N-morpholino)
ethanesulfonic
acid (MES) (pH 6.0 to 6.5 at 37#{176}C)
or Tris-MOPS
(pH 6.75 to 8.0
at 37#{176}C).
Fluxes were expressed
per liter of original cells to allow
comparison
of cells with different
MCHC.
Regulation
of cell volume
and cation
content.
In a first set of
experiments,
55 cells were treated with nystatin
to obtain cells with
increased
cation and water content. To study the effect of acid pH on
the volume regulatory
properties
of 55 cells, the cells were then
incubated
at 2% to 5% Hct in media containing
140 mmol/L
NaCI,
4 mmol/L
KCI, 1 mmol/L
phosphate,
I mmol/L
MgCI2,
10
mmol/L
Tris-MOPS,
pH 7.40 to 7.00 at 37#{176}C,
and 10 mmol/L
glucose.
To maintain
cell 2,3-diphosphoglycerate
(DPG)
constant
and avoid its depletion
in media with pH <7.4O,
adenine
and
inosine were used at concentrations
ofO.6 mmol/L
and 0.4 mmol/L,
respectively
(pH 7.40), and I .2 and 0.9 mmol/L,
respectively
(pH
7.0). ATP and 2,3-DPG
cell contents
were measured
with commercial kits (Sigma; ultraviolet
determination
with the phosphoglyceric
phosphokinase
and glyceraldehyde
phosphate
dehydrogenase
method). After eight- to 12-hour incubation
in a shaking
waterbath
at
37#{176}C,
cells were washed four times with choline washing
solution,
and Na and K content,
MCHC,
and phthalate
density distribution
were measured.
The pH of the medium was checked at the end of the
incubation
and was stable.
In another
set of experiments,
55 cells were first separated
according
to density. 55 cells were layered on top of discontinuous
Stractan
II density gradients
and prepared
according
to the specifications ofClark
et al3#{176}
The densities
used were 1.096, 1.101, 1.110,
1.115 g/mL, with a dense cushion of 1.150 g/mL (values at 25#{176}C).
The tubes were then spun at 25,000 rpm for 50 minutes.
Three
fractions
were collected
from the density-separated
S5 cells: the top
fraction,
containing
most of the reticulocytes,
the middle fraction
with the mature discoid cells, and the bottom fraction,
with mainly
BRUGNARA,
TOSTESON
HA, AND
ISC. The different
fractions
were then washed with choline washing
solution.
One aliquot of cells from each fraction
was incubated
in
isosmotic
media
at pH 7.40 and 7.00 for 8 to 12 hours. The
remaining
cells from the three fractions
were treated with nystatin,
to obtain similar cation and water contents,
and then incubated
in
isosmotic
media at pH 7.40 and 7.0. Cell Na and K contents,
MCHC,
and phthalate
density profile were measured
at the beginning and end of incubation.
Chemicals.
NaCI and KC1 were purchased
from Mallinckrodt,
St Louis. Tris(hydroxymethyl)aminomethane
(Tris), albumin
(bovine, fraction
V), MOPS,
MES, HEPES,
ouabain,
sucrose,
and
nystatin were purchased
from Sigma, St Louis. Choline chloride was
purchased
from Calbiochem-Behring,
La Jolla, CA. MgC12 was
purchased
from Fisher Scientific,
Springfield,
NJ. Bumetanide
was
a gift from Leo Pharmaceutical,
Ballerup,
Denmark.
All chemicals
were reagent
grade, and all solutions
were prepared
using doubledistilled water.
RESULTS
Effect
of acid
cells.
pH
To study
and
cell
volume
the combined
on K efflux
effect
of varying
and volume
on K transport,
different
MCHCs
and Na
55
and
hemoglobin
different
K effiux
was
(0.1 mmol/L)
(0.01
basis
(Fig
1).
with ouabain
pH,
mmol/L)
Na-K-Cl
added
cells were
K contents,
to inhibit
cotransport
systems,
the
from
both
SS
cell
pH
modified
to have
on a mmol/kg
then
measured
and bumetanide
Na-K
pump
respectively.
at
and
As shown
the
in Fig
I, K effiux
from
SS cells was pH dependent,
with a pH
optimum
-6.7
to 7.0 and inhibition
at more alkaline
or acid
pH,
and
was
increased
Moreover,
also
volume
dependent
since
K effiux
in swollen
cells and reduced
in shrunken
cell swelling
induced
a marked
change
dependence
of K effiux.
The
bell-shaped
was
55 cells.
in the pH
dependence
on pH
noted in SS cells with normal
volume
was lost in swollen
55
cells since, under these conditions,
alkaline
pH did not inhibit
K effiux.
In these
experiments,
volume
was varied
by the
nystatin
300
method
and
the
osmolarity
mosm in all cases.
In another
set of experiments,
cells
with
different
volumes
larity
of the flux
(MCHC
32 g/dL).
bell-shaped
with
swelling.
previous
fluxes,’92’
port
cells.
density
two
To study
distribution
measured
pH
decreasing
the
was
sets
the
on
effect
of AA
solution
further
was
in
osmo-
the
density
but still
7.40
the
and
K
cotranspersists
distribution
at different
the cells
at pH
of
of KC1
of lowering
control
cells,
by
together
nature
dependence
tion was measured.
RBCs
incubated
because
of the titration
of negatively
stimulated
of experiments,
cell volume
were incubated
end of incubation,
washing
was
by varying
defining
the
cells.
of acidification
normal
donors
37#{176}C.At the
choline
that
with
K effiux
on pH and
research
decreases
shrunken
Effect
medium
produced
These
indicate
flux
medium,
in nystatin-treated
55 cells
As shown
in Fig 1, K effiux
had a
dependence
hypotonic
of the
of
in
AA
external
pH on
fresh
cells
from
pH for 1 2 hours at
were washed
with
the density
in acid
charged
distribu-
media
residues
swell
and
the compensatory
inward
movement
of chloride
and water.
However,
since in our experiment
the cells were washed
at
pH 7.40 after incubation,
this mechanism
was not responsible
for
the
volume
changes
observed.
As
shown
in Fig
2A,
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
EFFECT
OF ACID
PH ON
SICKLE
CELL
MCHC
489
DENSITY
(g/dL)
Osmolarity
(mosM)
.
0
#{149}
215
o 250
C
0
#{163}
30
o 300
#{163}
350
I 400
15
-C
a,
a,
C)
a’
20
Ala
0
E
E
1O
a)
+
0
0
6.0
7.0
8.0
6.0
External
pH
External
8.0
7.0
pH
Fig 1 .
Dependence
on external
pH of K effiux from CO-treated
SS cells. SS cells were treated
with nystatin
to have different
MCHC
and K content
(mmol/kg
Hb) (left). K contents
were 440. 355. 302. 255, and 227 mmol/kg
Hb in cells with MCHC of26.1.
30.4. 32.7. 35.2.
and 37.4 g/dL. respectively.
55 cells from another
patient
were treated
with nystatin
to have an MCHC of 32.6 g/dI (cell Na 27 mmol/kg
Hb, cell K 306 mmol/kg
Hb) (right) and incubated
in media with different
pH and osmolarities.
Flux media contained
140 mmol/L
NaCI (left)
or 100 mmol/L
NaCl and 0 to 100 mmol/L
choline chloride
(right).
1 mmol/L
MgCl2, 10 mmol/L
glucose.
0.1 mmol/L
ouabain,
0.01 mmol/L
bumetanide
and 10 mmol/L
Tris-MES
(pH 6.0 to 6.5 at 37’C) or Tris-MOPS
(pH 6.75 to 8.0 at 37’C).
Data are from one experiment
representative
of two experiments
in different
subjects.
I
Fresh
0
12 hours,
cells
pH 7.00
C
1 2 hours,
pH
7.40
100
Cl)
Cl,
a,
a,
C.,
C)
a,
C)
C
Cl)
C
a,
a)
A#{176}
Fig 2.
Effect
of pH on density
distribution
1.100
1.100
Density
Density
of fresh
AA cells.
(A) Control;
(B) + adenine
and inosine.
1.120
Fresh
AA cells
(MCHC
33.6
g/dL;
cell Na 21 mmol/kg
Hb; cell K 268 mmol/kg
Hb) were incubated
for 12 hours at 37’C in a medium
containing
140 mmol/L
NaCI. 3 mmol/L
KCI. 1 mmol/L
MgCl2.
1 mmol/L
K-phosphate.
10 mmol/L
Tris-MOPS.
pH 7.40 or 7.00. 10 mmol/L
glucose.
(B) media also contained
adenine
and inosine.
1 .2 and 0.9 mmol/L.
respectively.
at pH 7.0. and 0.6 and 0.45 mmol/L.
respectively.
at pH 7.40. After incubation.
the
erythrocytes
were washed
five times with choline washing
solution.
pH 7.40. at 4”C. Cell 2.3-DPG
content
was 16.7 mmol/kg
Hb in fresh
cells. 7.7 and 0.1 mmol/kg
Hb after incubation
with glucose alone at pH 7.40 and 7.0. respectively.
and 22.9 and 18.4 mmol/kg
Hb after
incubation
with glucose
plus adenine
and inosine
at pH 7.40 and 7.0. respectively.
Data
are from
one experiment
representative
of two
experiments
in different
subjects.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
490
BRUGNARA.
AA
cells
swell
2,3-DPG
when
content
media.29
inward
As
concentration
ing AA
and
at
pH
<7.40
reduced
cells
with
of
on density
out effects
at pH
next
Figure
3 shows
fewer
dense
cells
of dense
studies
2B,
when
the
constant
by incubat-
adenine,
AA
maintained
cells
incubated
in acid
on
the
density
effect
of
first
studied
experiments
and
cells.
were
in
in a patient
another
Exposure
incubation
performed
in fresh
patient
with
with
in SS
7.40
cells
had
volume.
incubated
resembled
pH
that
method.
the
lowered
At
for
in Fig 4, 55
When
progressively
occurred.
nystatin
SS
swollen
The
SS cells,
experiments
inhibitors
the
of fresh
Thus,
pH
SS cells
of
However,
the
dense
55
(inhibitor
relatively
ouabain
and
transport,
at pH
little
467
or no
by the
Na/K
progressive
cells
of
0.01
mmol/kg
Hb)
(initial
contained
per kilogram
of Hb without
Na
mmol
268
Indeed,
SS cells
K per
can
cell
20 mmol
transport
kilogram
not
the
after
without
affected
or
Na-K-Cl
and
and
inhibitors
of Hb
by
mmol/L)
12-hour
Na
Na
be
cotransport.
0.1
of
by
deoxygenation.
also in nystatin-
was
pump,
(inhibitor
mmol/L).
swollen
and
Na/K
of
swelling
formation
Na-K-Cl
formation
the
bumetanide
7.40
and
cell
distribution
transport
inhibitor.
above were performed
pump
back
incubation
before
cell
in an
shrunk
of the
density
dense
TOSTESON
1 2 hours
cells
to 7.00,
7.00,
without
any
described
ouabain
a sizable
swollen
then
As shown
original
was
cells
formation
to pH 7.1 induced
at pH
the
medium
the
pH
untreated
toward
and
medium.
achieved
without
external
Ca and without
This process
occurs
in fresh 55 cells and
SS
external
technique
isosmotic
55
medium
distribution
of lowering
nystatin
shrinkage
2,3-DPG
relatively
being
was
of dense cells, whereas
effect on cell density.
Other
induces
a sizable
to maintain
elec-
and cation
content
of 55 cells. To rule
to use of the ionophore
nystatin,
the
pH
blood.
the
in acid
7.40.
studied
distribution
secondary
of acid
fraction
Fig
and
after
acidification
We
influence
inosine
volume
washed
Effect
cells.
in
was maintained
normal
then
shown
because
by incubation
The decrease
of cell 2,3-DPG
movement
of chloride
and water
troneutrality.
their
incubated
is markedly
HA, AND
with
co-
incubation
K
37 and
=
324
mmol
and
75 mmol
ouabain
K
and
100
Cl)
0)
0
a)
C,)
C
50
1)
A
0
1.080
1.100
1.120
1.136
Density
100
80
C,,
w
U
60
L
a,
Cl,
40
a,
20
B
0
1.06
1.08
1.10
Density
1.12
1.14
Fig 3.
Effect
of pH on density
distribution
of
fresh SS cells. Fresh 55 cells were
incubated
for
12 hours
at 37’C
in a medium
containing
140
mmol/L
NaCI, 3 mmol/L
KCI. 1 mmol/L
MgCI2.
1
mmol/L
K-phosphate.
10 mmol/L
Tris-MOPS.
pH
7.40 or 7.10. and 10 mmol/L
glucose.
Media
also
contained
adenine
and inosine.
1 .2 and 0.9 mmol/
L. respectively.
at pH 7.0. and 0.6 and 0.45 mmol/
L. respectively.
at pH 7.40. After
incubation.
the
erythrocytes
were washed
five times with choline
washing
solution,
pH 7.40. at 4’C. Two different
patients
are shown
(A and B) (A) Total
cation
content
at the beginning
of incubation
was 378
mmol/kg
Hb (MCHC
30.3 g/dL.
K content
351
mmol/kg
Hb) and decreased
after 12 hours to 337
mmol/kg
Hb (MCHC
31.2
g/dL.
K content
315
mmol/kg
Hb) at pH 7.40 and to 269 mmol/kg
Hb
(MCHC
33.2 g/dL. K content
237 mmol/kg
Hb) at
pH 7.10. D,, and R,, were 1 .100 and 0.010 in fresh
cells. 1.100 and 0.008
at pH 7.40. and 1.108 and
0.01 6 at pH 7.1 0. respectively.
(B) Total
cation
content
at the beginning
of incubation
was 243
mmol/kg
Hb (MCHC
36 g/dL.
K content
184
mmol/kg
Hb) and decreased
after 12 hours to 237
mmol/kg
Hb (MCHC
36.1
g/dL.
K content
182
mmol/kg
Hb) at pH 7.40 and to 166 mmol/kg
Hb
(MCHC
41.8 g/dL. K content
116 mmol/kg
Hb) at
pH 7.1 0. D,, and R, were 1 .1 1 6 and 0.036 in fresh
cells. 1.114 and 0.022
at pH 7.40. and 1.124 and
0.022
at pH 7.10.
respectively.
Similar
results
were obtained
in two other patients.
(A): (U). fresh
cells; (0). 12 hours. pH 7.10; (#{149}).
12 hours, pH 7.40.
(B): (#{149}).
fresh cells; (0). 12 hours. pH 7.10; (U). 12
hours.
pH 7.40.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
EFFECT
OF ACID
PH ON
SICKLE
CELL
DENSITY
491
100
Cl)
e
0)
0
a)
Cl)
C
a)
0
1.100
Density
Fig 4.
Effect of external
pH on density
distribution
of swollen
SS cells. SS cells were swollen
with
Na and K content
34 and 410 mmol/kg
Hb. respectively)
and then incubated
for 12 hours at 37’C
g/dL.
different
pH.
with
composition
similar
to that
detailed
in the
legend
to Fig
2. At
the
end
the nystatin
technique
(MCHC
27.1
(2% Hct) in an isosmotic
medium
at
of incubation.
the
erythrocytes
were
washed
five
times with choline washing
solution
and the phthalate
density
distribution
was measured.
D,0 and R,, were 1.103 and 0.025 in fresh cells,
1.080 and 0.009 in nystatin-swollen
cells at zero time. 1.087 and 0.015 at pH 7.40. 1.096 and 0.015 at pH 7.30. 1.096 and 0.018 at pH 7.20.
1.098 and 0.024 at pH 7.10, and 1.097 and 0.030 at pH 7.00. respectively.
Data are from one experiment
representative
of two experiments
in different
subjects.
bumetanide.
After
incubation
at
contents
were 28 and 28 1 mmol/kg
transport
inhibitors,
tively,
with
Effect
cells
and
ouabain
of
acid
pH
separated
variations
experiments
to density
and bottom
and
in Fig
Similar
5, the
to
were
patients
dense
cells
fractions
middle
the top
fraction),
(Fig
at pH
2,3-DPG
of whole
55
in the three
that
absent
and
cells
7.40
acid
pH-induced
the
top
in the
was
range
and
7.00,
bottom
pH
accompanied
(R;
middle
density
by acid
at pH
from
are
heterogeneous
fraction
in the
0.006
to 0.013
response
and
then
obtained
when erythrocytes
were first swollen
with
exposed
to pH 7.40
at pH
7.10.
Swollen
55
cells
and
density
at pH
from
7.00
to acid
pH
fraction
by
acid
(data
pH
in
nystatin-swollen
not shown).
pH can
tory
that
effect
on K loss
cotransport,
directed
it should
K gradient
concentration.
density
fraction
ing 140 mmol/L
cells
a large
swelling
The
abolished
is abolished
increased
K content,
be
cell volume
suggesting
by acid
the K gradient
is inwardly
directed
K gradient
is the
induced
but
the
since
wardly
To
system
pH,
is due
that
by acid
mediates
dense
and 7.0,
constant.
hypothesis
for this
system
and
densest
the
least
and
the external
cotransport
K
K, Cl,
and
of the
is acti-
water
gain
directed.
Thus,
the outdriving
force for the net K
that the KC1
cell shrinkage
experiments
We previously
is present
only
densest
fractions
were
cotransport
induced
fraction
fraction
is markedly
shrink
in the
least
dense
RBC population)
AA cells from the
incubated
different.
when
by
in AA cells sepashowed
that the
at pH
with adenine
and inosine,
to maintain
As shown
in Fig 7, the behavior
of the
dense
by
outwardly
pH.
test further
the
is responsible
cotransport
net
least
the
whereas
to an increase
KC1
density
of
water
and middle
the stimula-
the
by increasing
vated
loss
as
fraction
of AA cells (2% to 5% of the entire
and is nonactive
in the densest
fraction.22
did
was
K, Cl, and
is abolished,
shrunk
became
they
cells
of
Figure
6 shows
that when cells from the top
were incubated
at pH 7.0 in media
containKC1, the RBCs
did not shrink,
but actually
KC1
bottom
55 cells
reduced
in 55 erythrocytes
As a consequence,
cell
induce
by cell
for
1 2 hours
that
of acid pH is diminished
but not completely
abolished.
If the cell shrinkage
induced
by acid pH is mediated
acidification,
we performed
rated
according
to density.
fraction
had a markedly
different
behavior,
since
not shrink
at pH 7.40. However,
formation
of dense
induced
and/or
is greatly
fraction.’9
from the
the nystatin
fractions
7.40 and
the
acid
cell
middle
den-
on the basis
loss and cell shrinkage
in 55 cells of the top
density
fractions.
In cells of the bottom
fraction,
swelled.
of
be explained
swelling
and
for
can
system
density
(Fig
5).
technique
bottom
top
by an increase
from
in their
at 37#{176}C.
Cells of the top and middle
back
toward
the original
volume
still
of
density
results
of this transport
of the bottom
KCI
7.0.
the
formation
and
These
the top and middle
density
fractions
have
a very
active
volume
and pH-dependent
KCI cotransport,
whereas
activity
with
As shown
fraction,
and from 0.006
to 0.016
for the
suggesting
that these relatively
homogeneous
fractions
AA
separated
blood increased
density
fractions
in
density
Similar
results
were
three
density
fractions
denser
SS
depletion.
the
fractions
60%
respec-
to the marked
with sickle
cell
in 55
media
in D50 induced
density
middle
sity
was
Due
of patients
performed
maximal
increase
of
density.
to avoid
showed
and
The
the
inosine,
was
Hb,
(Stractan
II).#{176}Whole
55 blood
top,
density
fractions
were incubated
for I 2
density
experiments
same
mmol/Kg
distribution
in RBCs
at 37#{176}C
in plasmalike
adenine
5).
density
according
according
middle,
230
7.0, the Na and K
respectively,
without
bumetanide.
on
in density
disease,
hours
79 and
and
pH
Hb,
exposed
7.40
2,3-DPG
least dense
Erythrocytes
to pH
of
<7.40.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
492
BRUGNARA,
A. Whole
#{149}
Fresh
blood
12
S 12
cells
hours,
hours,
0
C.
pH 7.00
pH 7.40
Middle
HA, AND
TOSTESON
fraction
100
Cl)
0,
a,
a,
0
0
a,
Cl)
C
a,
0
50
0
0
1.080
1.100
1.120
1.136
1.080
1.100
1.120
B. Top
1.136
Density
Density
fraction
D. Bottom
fraction
100
Cl)
Cl)
C)
a,
Cl)
C
a,
0
50
0
0
1.080
1.100
1.120
1.136
1.100
1.060
1.120
1.136
Density
Density
Fig 5.
Effect of pH on density
distribution
of whole SS blood and three density
fractions
of 55 cells. Stractan
density
gradients
were
used to isolate top (MCHC
31 .4 g/dL; K content
301 mmol/kg
Hb; Di,, 1 .097; R,, 0.006).
middle (MCHC
34 g/dL; K content
290 mmol/kg
Hb;
D 1 .102; R,, 0.006).
and bottom
(MCHC
40 g/dL; K content
1 89 mmol/kg
Hb; Di,, 1 .1 16; R 0.019)
fractions
of SS cells (MCHC
36 g/dL; K
content
261 mmol/kg
Hb; D,,, 1 .102; R,, 0.012).
Unseparated
SS cells (left top panel) and the three density
fractions
were subsequently
incubated
for 12 hours at 37’C in a medium
containing
140 mmol/L
NaCI. 3 mmol/L
KCI. 1 mmol/L
MgCI2.
1 mmol/L
K-phosphate.
10
mmol/L
Tris-MOPS,
pH 7.40 or 7.00. 10 mmol/L
glucose.
The media also contained
adenine
and inosine.
1 .2 and 0.9 mmol/L.
respectively.
at pH 7.0. and 0.6 and 0.45 mmol/L.
respectively.
at pH 7.40. The phthalate
density
profile was measured
at the beginning
and end of
incubation
after the cells were washed
four times with choline washing
solution.
Final K content
in unseparated
SS cells, top, middle, and
bottom
density
fractions
were 261. 299. 291 #{149}and 202 mmol/kg
Hb, respectively,
at pH 7.40. and 188. 218. 190. and 140 mmol/kg
Hb.
respectively,
at pH 7.00. Data are from one experiment
representative
of three experiments
in different
subjects.
Cells
similar
from
pH.
support
the densest
fraction
swell
when
These experiments
in density-separated
the
hypothesis
transport
system
formation
of dense
that
induced
SS and
K
loss
by
pH
AA
cells.
exposed
AA
through
<7.40
the
to a
cells
KC1
co-
is responsible
for
We showed
that
dense
cells
is due
our
or pH values
responsible
experimental
formation
induced
cells
of K, Cl, and
system
<7.40
water.
Since
is greatly
(Fig
1),1927
of dense
a conclusion
of dense
cells
in the medium
can be induced
is in the fully ligated
<7.40.
The formation
for formation
conditions,
fact that formation
the K concentration
cell
of dense
the KC1 cotransport
swelling
is probably
to a loss
fraction
In mature
normal
to pH
this
of
exposure
K
RBC
pathway
SS cells
supported
by acidification
is also
found
acid
To
in the
containing
cell
by two
the net
hemoglobin
pH induces
compensate
for
cell swells.
acidification
These
and shrinkage
on
when
deoxygenated
Canessa
mechanisms
of mature
AA
acidification
in media
et a127 showed
that
which
cells
negative
and
an
fracA,
different
charge
of
2,3-DPG,
inward
movement
of
Second,
long-term
a decrease
the
in 2,3-DPG
decrease
promote
are
possess
densest
hemoglobin
swelling
polyvalent
nonpermeant
negatively
charged
valent
chloride
and hence
water
move into
under
in the
RBCs
induces
which
not
a compensatory
with cell swelling.
to relatively
content.29
by the
is prevented
by increasing
(Fig 6). Moreover,
dense
and
First, by decreasing
solutes
such
as
acidification
induces
chloride
and water,
stimulated
erythrocytes,
human
<7.40
in
state
net
of AA
cotransport
system,
does not (Fig 7).
mechanisms.
nonpermeant
when hemoglobin
in media
with pH
loss through
by cell
formation
dense
exposure
DISCUSSION
55 erythrocytes
by incubation
least
active
KC1
tion, which
overcome
residue,
the cell
in
this
monoand the
cell swelling
on
by KC1
loss
of 55 cells. 55 cells shrink
with acid pH.3’ Research
by
27%
of the
cells
from
the
SS2
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
EFFECT
OF ACID
H
ON SICKLE
CELL
493
DENSITY
fraction
shrink
when exposed
7.0 with ouabain
added.
Our
100
formed
constant
80
being
Our
exposed
60
a,
fractions
exposed
K,
Cl,
active
40
and
with
cells
density
Our
tion
have
can
be found
days
after
appear
formed
Density
that
that
channel
and
cell
in the
have
Whether
by
cell
RBC
ISC
of
5). Studies
on
correspon-
age
and
that
as early
survival
of 55
acidification
can
of
59Fe
as ten
in 55
occur
the
cells
directly
cells
fractions
young
due
K
no studies
density
when
55
of 55
cells
become
to activation
occurs
in
from
Ca-dependent
Unfortunately,
of labeled
so that how and
to be established.
shrinkage
cotransport
SS cells
59Fe administra-
fraction
cotransport.
survival
(Fig
after
and
of
of dense
described
erythrocytes,
ISC remains
and
is very
are not the oldest
cells but are
Theoretic
work by Lew et al’5
activation
KC1
fresh
an approximate
densest
through
vivo
of KCI
is not
clear.
Certainly,
acidification
of the magnitude
used in our experiments
does not occur
in vivo. However,
we showed
(Fig 4)
that formation
of dense
cells is not an all-or-none
phenomenon,
are
but
progressively
acidified
actively
every
metabolizing
increases
time
they
tissues
dense
when
pass
and
the pH is <7.40.
through
the
hypoxic
0 time
and
RBCs
capillaries
acidotic
of
condi-
12 hours
pH 7.40
pH 7.00
#{149}
Densest
hours at 37’C in a medium
containing
140 mmol/L
NaCI, 3 mmol/L
KCI, 1 mmol/L
MgCI2,
1 mmol/L
K-phosphate,
10 mmol/L
Tris-MOPS,
pH 7.40 or
7.00.
1 0 mmol/L
glucose.
The media
also contamed
adenine
and inosine.
1 .2 and 0.9 mmol/L.
respectively.
at pH 7.0 and 0.6 and 0.45 mmol/L.
respectively.
at pH 7.40. In the least dense fraction.
MCHC
(g/dL)
and K content
(mmol/kg
Hb)
were 30.5 and 298 at zero time, 30 and 298 after
12 hours at pH 7.40, and 32.9 and 255 after
12
hours at pH 7.0. respectively.
In the densest
fraction, MCHC
(gIdL) and K content
(mmol/kg
hemoglobin)
were 37.9 and 218 at zero time. 37.9 and
218 after
12 hours at pH 7.40. and 37.5 and 206
after
1 2 hours at pH 7.0. respectively.
Data are
from one experiment
representative
of two experiments
in different
subjects.
only
Studies
volumewhich
that
fractions
density
bottom
the
system,
RBCs
formation
the
denser
have the largest
K loss and
external
pH as compared
denser
at best
labeling.’2
reticulocytes
times
and at
the top and
become
through
suggest
of normal
between
Least
Fig 7.
Effect
of pH on density
distribution
of
the least dense and densest
fractions
of AA cells.
Least dense fraction
(top 2%) and densest
fraction
(bottom
5%) were isolated
with Stractan
density
gradients.
The two fractions
were incubated
for 12
also
to indicate
that ISC
from young
cells.”4
suggests
Fig 6.
Effect of external
Na and K on density
profile
of SS cells
from top fraction.
Fresh SS cells from the top fraction
(MCHC
29.7
g/dL. Na 24 mmol/kg
Hb. K 365 mmol/kg
Hb) were incubated
for
12 hours at 37’C at pH 7.0 in media containing
either
130 mmol/L
NaCl. 10 mmol/L
KCI. or 130 mmol/L
KCl. 10 mmol/L
NaCI. Media
also contained
1 mmol/L
K-phosphate.
1 mmol/L
MgCl2.
10
mmol/L
Tris-MOPS.
1 .2 mmol/L
adenine.
0.9 mmol/L
inosine,
and
10 mmol/L
glucose.
Hct was 2%. After
incubation.
the erythrocytes were then washed
five times with choline washing
solution.
pH 7.40. at 4’C. Final MCHC.
Na. and K contents
were 36.9 g/dL.
37 mmol/kg
Hb, and 219 mmol/kg
Hb. respectively,
for cells
incubated
in NaCl medium
and 25.3 g/dL. 13 mmol/kg
Hb. and 478
mmol/kg
Hb. respectively,
for cells incubated
in KCI medium.
Data
are from one experiment
representative
of two experiments
in
different
subjects.
(-.4--).
Top fraction,
fresh cells; (-U-).
130
mmol/L
KCI, pH 7.0; (-0-).
130 mmol/L
NaCI. pH 7.0.
loss
data
the other
shown
exists
in
air to pH
were per-
5).
cotransport
fraction
by lowering
distribution
dence
4
from
(Fig
water
the top, reticulocyte-rich,
cell shrinkage
induced
20
incubation
all cells
erythrocytes
pH
KC1
in 55 cells.
at room
which
that the lack of cell swelling
in 55 cells
in the oxygenated
state is due mainly
to
chloride-dependent
C)
C
of 55
to acid
data indicate
to pH <7.40
a marked
I-
a,
density
after
a,
C)
without
ouabain
at longer
2,3-DPG,
indicate
that
middle
Cl)
for one hour
experiments,
U
0
I
0
(1)
a)
0
I.-
a)
Cl)
C
c3
0
1.080
1.100
1.120
Density
1.136
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
494
BRUGNARA.
tions
the
are
normally
renal
small
but
produce
an
found
medulla
and
in the
hypertonic
in conditions
repeated
changes
in
cell
a significant
loss of potassium
pH,
and
active
volume-
environment
of low blood
pH
may
content
of
flow.
These
in cells
having
KC1
co-
Previous
in vitro
focused
through
drating
studies
on
on changes
mediated
not require
pump
ation,
formation
induced
the Ca-activated
effect
of the Na
dehydration
does
Studies
K channel’#{176} or through
pump.”
As we have
by
external
the
Ca,
by Mohandas
that
the
and
K fluxes
cells.
KC1
In the
latter
formation
of
with
to
sickle
in
relative
The increased
cellular
fresh
55 cells
has
quences.’7”8
Hb, 2,3-DPG
present
Horiuchi
As
content
at a given
increases
oxygen
dehydration.
mechanism
water
Ca
be
cells.
and
essential
Asakura’#{176}
Na
rate
of
requisites
Other
investigators
of membrane
cell dehydration
oxidative
of sickle
for
have
damage
cells.’7”8
of
concentration
important
each
of 2,3-DPG
pathophysiologic
amount
tension.
has
Other
oxygen
to the
of
yet
to
be
Thus,
content
are
cytoplasmic
to its effect
water
across
on the
the cell
another
either
with
top-fraction
AA
be important
in stimulating
inducing
a larger
K loss
consequence
of 55 cells
and
of
is a reduction
directly
through
increased
formation
cells.
the
and
the
KCI
cell
increased
of cell volume
and
the compensatory
loss
affinity
of
tissues
and
of the
of Hb S polymer.
In summary,
we showed
induces
loss of KCI and water
attributed
to the volume-,
cotransport,
cells and
these effects
least dense
that
lowering
in 55 RBCs.
pH-,
which
is active
in the least dense
and
in most
fraction
the
This
internal
pH
effect can be
chloride-dependent
KCI
density
fractions
of normal
AA
take place in CO-treated
55
AA cells, they do not require
of 55
RBCs.
cells and
deoxygena-
tion and polymerization
of S Hb. However,
by inducing
dehydration
and increasing
Hb S concentration,
they
markedly
affect
observed
conse-
deoxyhemoglobin
consequences
cells
due
H, and
as compared
cell pH could
system
and
content,
Since
normal
mechanisms
for RBC dehycertain
conditions
in sickle
importance
the
of 55
a slow
55
TOSTESON
of chloride
and water
or indirectly
through
activation
of the
KC1 cotransport
due to the lower cell pH. Both mechanisms
contribute
to cell dehydration,
increased
Hb concentration,
deoxygen-
and
2,3-DPG
of
shrinkage,
of Cl,
AND
shown
by Kaperonis
et al,’9 the increased
is responsible
for the lower internal
pH of
55 cells
This lower
cotransport
the dehyshown,
cell
distortion
Due to its effect
in lowering
facilitates
oxygen
delivery
concomitantly
either
does not require
cells.
external
Any one of the abovementioned
dration
may be operative
under
cells
and the
determined.
cells
or not the Na-K
morphologic
shown
importance
K loss and
55
of deoxygenation-induced
report,
dense
the
whether
Ct alM and
were
emphasized
in determining
cotransport
occurs
magnitude
increases
deoxygenation
of dense
by deoxygenation,
is inhibited
by ouabain,
and
since it occurs
in CO-treated
showed
membrane.
2,3-DPG
content
and cell
distribution
top-fraction
transport.
have
2,3-DPG
acidification
equilibrium
eventually
chloride-dependent
increased
HA,
Hb S polymer
formation
and
cell
in
cell
can
sickling.
ACKNOWLEDGMENT
We are grateful
Center
at Boston
study. The support,
greatly appreciated.
to Dr Lilian McMahon
of the Boston Sickle Cell
University
for providing
blood samples
for our
advice, and criticism of Dr H. Franklin
Bunn are
REFERENCES
1. Fabry ME, Nagel RL: Heterogeneity
of red cells in the sickler:
A characteristic
with practical
clinical and pathophysiological
implications. Blood Cells 8:9, 1982
2. Mohandas
N, Phillips WM, Bessis M: Red blood cell deformability and hemolytic
anemias.
Semin Hematol
16:95, 1979
3. Lawrence
C, Fabry
ME, Nagel
RL: Red cell distribution
width parallels
dense red cell disappearance
during painful crises in
sickle cell anemia.
J Lab Clin Med 105:706, 1985
4. Eaton WA, Hofrichter
J: Hemoglobin
S gelation and sickle cell
disease. Blood 70:1 245, 1987
5. Gardos G: The permeability
of human erythrocytes
to potassium. Acta Physiol Acad Sd Hung 10:185, 1956
6. Glader BE, Nathan
DG: Cation permeability
alterations
during sickling: Relationship
to cation composition
and cellular
hydration of irreversibly
sickled cells. Blood 5 1:983, 1978
7. Jensen M, Shohet SB, Nathan
DO: The role of red cell energy
metabolism
in the generation
of irreversibly
sickled cells in vitro.
Blood 42:835, 1973
8. Bookchin
RM, Ortiz OE, Lew VL: Red cell calcium transport
and mechanisms
ofdehydration
in sickle cell anaemia,
in Beuzard Y,
Charache
5, Galacteros
F (eds): Approaches
to the Therapy
of
Sickle Cell Anemia.
Paris, Les Editions
INSERM,
1986, p 291
9. Ohnishi
ST. Horiuchi
KY, Horiuchi
K: The mechanism
of in
vitro formation
of irreversibly
sickled cells and modes of action of its
inhibitors.
Biochem Biophys Acta 886:1 19, 1986
10. Horiuchi
K, Asakura
T: Formation
of light irreversibly
sickled cells during deoxygenation-oxygenation
cycles. J Lab Clin
Med 110:653, 1987
I 1 . Clark MR. Guatelli
JC, White AT, Shohet SB: Study on the
dehydrating
effect of the red cell Na/K
pump in nystatin
treated
cells with varying Na and water contents.
Biochim
Biophys Acta
646:422, 198!
12. Joiner CH, Platt OS, Lux SE IV: Cation depletion
by the
sodium pump in red cells with pathological
cation leaks. Sickle cells
and xerocytes.
J Clin Invest 78:1487,
1986
13. Izumo H, Lear 5, Williams
M, Rosa R, Epstein FH: Sodiumpotassium
pump, ion fluxes, and cellular
dehydration
in sickle cell
anemia.
J Clin Invest 79:1621,
1987
14. Tosteson
DC, Shea E, Darling RC: Potassium
and sodium of
red blood cells in sickle cell anemia.
J Clin Invest 3 1 :406, 19S2
IS. Tosteson
DC, Carlsen E, Dunham
ET: The effects of sickling
on ion transport.
I. Effect of sickling on potassium
transport.
J Gen
Physiol 39:31, 1985
16. Berkowitz
LR, Orringer
EP: Passive sodium and potassium
movements
in sickle erythrocytes.
Am J Physiol 249:C208,
1985
17. Hebbel
RP, Eaton
JW, Balasingam
M, Steinberg
MH:
Spontaneous
oxygen radical generation
by sickle erythrocytes.
J Clin
Invest 70:1253, 1982
18. Ney PA, Hebbel RP: Enhanced
RBC potassium
leak due to
synergism
between
membrane
oxidation
and deformation:
Insight
into sickle RBC dehydration.
Blood 70:66a, 1987 (abstr)
19. Brugnara
C, Bunn HF, Tosteson
DC: Regulation
of erythrocyte cation and water content in sickle cell anemia. Science 232:388,
1986
20. Brugnara
C, Tosteson
DC: Inhibition
of K transport
by
divalent cations in sickle erythrocytes.
Blood 70:1810,
1987
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
EFFECT
OF ACID
PH ON SICKLE
CELL
DENSITY
21 . Brugnara
C, Kopin AK, Bunn HF, Tosteson
DC: Regulation
of cation content and cell volume in erythrocytes
from patients with
homozygous
hemoglobin
C disease. J Clin Invest 75:1608,
1985
22. Brugnara
C, Tosteson
DC: Cell volume, K transport
and cell
density in human erythrocytes.
Am J Physiol 252:C269,
1987
23. Hall AC, Ellory JC: Evidence
for the presence
of volumesensitive
KCI cotransport
in young
human
red cells. Biochem
Biophys Acta 858:317,
1986
24. Lauf PK: Thiol-dependent
passive K/Cl transport
in sheep
red cells: II. Loss of Cl
and N-ethylmaleimide
sensitivity
in
maturing
high K cells. J Membrane
Biol 73:247, 1983
25. Brugnara
C, Bunn HF, Tosteson
DC: Ion content and transport and the regulation
of volume in sickle cells. Ann NY Acad Sci
(in press)
26. Canessa
M, Spalvins
A, Nagel RL: Volume-dependent
and
NEM-stimulated
K, Cl transport
is elevated
in SS, SC and CC
human red cells. FEBS Lett 200:197,
1986
27. Canessa
M, Fabry ME, Blumenfeld
N, Nagel RL: Volumestimulated,
Cl-dependent
K efflux is highly expressed
in young
human red cells containing
normal hemoglobin
or HbS. J Membrane
Biol 97:97, 1987
28. Rodgers GP, Schechter
AN, Noguchi
CT: Cell heterogeneity
in sickle cell disease: Quantitation
of the erythrocyte
density profile.
J LabClin Med 106:30, 1985
29. Deuticke
B, Duhm J, Dierkesmann
R: Maximal
elevation
of
2,3-Diphosphoglycerate
concentrations
in human
erythrocytes:
Influence
on glycolytic
metabolism
and intracellular
pH. Pflugers
Arch 326:15, 1971
495
30. Clark MR. Morrison
CE, Shohet
SB: Monovalent
cation
transport
in irreversibly
sickled cells. J Clin Invest 62:329,
1978
31. Fabry ME, Nagel RL: The effect ofdeoxygenation
on red cell
density: Significance
for the pathophysiology
of sickle cell anemia.
Blood60:1370,
1982
32. Luthra
MG, Friedman
JM, Sears DA: Studies
of density
fractions
of normal human erythrocytcs
labeled with iron-59 in vivo.
J Lab Clin Med 94:879, 1979
33. McCurdy
PR, Sherman
AS: Irreversibly
sickled cells and red
cell survival in sickle cell anemia. Am J Med 64:253, 1978
34. Bertles iF, Milner PFA: Irreversibly
sickled erythrocytes:
A
consequence
of the heterogeneous
distribution
of hemoglobin
types
in sickle cell anemia. J Clin Invest 47:1731,
1968
35. Lew VL, Freeman
Ci, Ortiz OE, Bookchin
RM: A new
hypothesis
on the origin of irreversibly
sickled cells (ISCs):
Predictions from an integrated
reticulocyte
model.
Clin Rca 36:415a,
I 988
36. Mohandas
N, Rossi ME, Clark MR:
Association
between
morphologic
distortion
of sickle cells and deoxygenation-induced
cation permeability
increase.
Blood 68:450, 1986
37. Seakins
M, Gibbs WN, Milner PF, Bertles
iF:
Erythrocyte
Hb-S concentration.
An important
factor in the low oxygen affinity
of blood in sickle cell anemia. J Clin Invest 52:422, 1973
38. Bunn HF, Forget BG: Hemoglobin:
Molecular,
Genetic
and
Clinical Aspects.
Philadelphia,
Saunders,
1986
39. Kaperonis
AA, Bertles
iF,
Chien S: Variability
of intracellular pH within individual
populations
ofSS and AA erythrocytes.
Br
J Haematol
43:391, 1979
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
1989 74: 487-495
Acid pH induces formation of dense cells in sickle erythrocytes
C Brugnara, T Van Ha and DC Tosteson
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