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2,3-Diphosphoglycerate
as
Content
a Function
of
Normal
By
A
STAvnos
parallel
cerate
HAIDAS,
decline
of
(2,3-DPG)
cular
hemoglobin
cells
during
and
2,3-DPG
depletion
T
EFFECT
HE
oxygen
livery
of red
It has been
in vivo
as
red
blood
cells.8
and
in
vivo
simultaneous
with
KAPLAN
to restore,
of
inosine
in senescent
young
2,3-DPG
is
cells
and
More
aged
study
our
to
found
compared
results
restore
with
as compared
cells.
(2,3-DPG)
of
age
of
be
in
vitro
working
in
of heparinized
the
laboratory.
both
has
aged
2,3-DPG
not
such
a study.
and
oxygen
been
in
the
de-
curve
of
and also
in
was
affinity
also
in red
reported.
Furthermore,
incubation
different
upon
oxygen
of 2,3-DPG
previously
upon
significantly
MATERIALS
ml
recently
a decline
red blood
cells.9
2,3-DPG
to
in
significantly
stor-
of different
cells
Twenty-four
sexes
capacity
presence
impaired
After
vitro
the
the
2,3-DIPHOSPHOGLYCERATE
blood
here
red
vestigated,
in
in
JEAN-CLAUDE
AND
age,
aging.
to
vitro
fractions
present
depleted
cells
red
in
the
cell
We
due
OF
in old
However
in
Age
of hemoglobin
and consequently
upon
the
cells to tissues
is now well established.46
found
that a shift to the left of the oxygen
dissociation
hemoglobin
occurs
in ACD
blood
during
storage,7
in
blood
LAmE,
of intracorpus-
vivo
Cell
Affinity
affinity
intracorpuscular
described
in
Oxygen
Individuals
2,3-diphosphogly-
is found
their
Red
DoIINIQuE
P50
and
the
capacity
with
inosine
vivo
aged
of
was
red
in-
blood
cells.
AND
venous
blood
Separation
of
METHODS
was
red
obtained
blood
cells
from
normal
adults
into
different
age
of both
fractions
was
performed
by centrifugation
in mixtures
of phthalate
esters of different
specific
gravity
according
to the method
described
by Danon
et al.10’ll
After removal
of leukocytes
and reticulocytes
in a preliminary
step, four fractions
were
prepared:
(1) Light
or “young”
red blood
cells corresponding
to the top fraction
(10%
of
total).
(2) Dense
or “old” red blood
cells corresponding
to the bottom
fraction
(10%
of
total).
(3)
Intermediate
red blood
cells
corresponding
to the middle
fraction
(20%
of total).
The light fraction
contained
less than 1.5% reticulocytes.
The other fractions
were
devoid
of reticulocytes.
(4)
Total
samples
of unfractioned
red blood
cells
were
also
From l’Institut
de Pathologie
Mol#{233}culaire, Paris, France.
Submitted
April 5, 1971; revised
May 11, 1971; accepted
Mat, 16, 1971.
Supported
in part by Grant
67.00.564
from
the D#{233}l#{233}gation
G#{233}n#{233}rale
4 la Recherche
Scientifique
et Technique.
STAvnos
HAIDAS,
M.D.:
Assistant,
Service
de Pediatric,
Clinique
Universitaire,
H#{244}pital
Sainte-Sophie,
Athens,
Greece;
Visiting
Research
Assistant,
Institut
National
de la Sante de
Recherche
M#{233}dicale, Paris, France.
DoMINIQuE
LABIE,
M.D.,
PH.D.:
Director
of Research,
Institut
National
de liz Sante de la Recherche
Medicale,
Institut
de Pathologic
Mol#{233}cislaire,
Paris,
France.
JEAN-CruDE
KAPLAN,
M.D.:
Professeur
Agr#{233}g#{233}
de Biochimie
M#{233}dicale,
Institut
de Pathologic
Moleculaire,
Paris, France.
BLOOD,
VOL.
38,
No.
4
(OCTOBER),
1971
463
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464
HAmAs,
LABIE,
AND
KAPLAN
40
Oxygen
affinity
P50
-35
::
20
P50 in red cell fractions
different
age
obtained
2 3 - D PG
10 normal
,
young;
individuals.
0,
mediate;
D
of
in
old;
I,
Y,
inter-
T, total.
10
‘:
0
submitted
to the same
stress
as the other
fractions
by passing
whole
blood
through
a
phthalate
ester mixture.
All the red blood
cell fractions
(total,
light,
dense,
and
intermediate)
were
then
thoroughly
washed
in saline
to remove
any
contaminating
phthalate
esters.
The quality
of the separation
was verified
by measuring
the activity
of glucose-6-phosphate
dehydrogenase
(C6PD)
and hexokinase
(HK)
in light
and dense
fractions.
C6PD
activity
was
1.5 and HK activity
2.5-fold
greater
in the light
fractions.
Oxygen
equilibrium
curves
were
determined
spectrophotometrically
in a Unicam
SP
800 spectrophotometer,
according
to
the technique
of Benesch
et al.’2
modified
by
Bellingham
and
Huehns.’3
Measurements
were
performed
on washed
red blood
cell
fractions
suspended
in isotonic
phosphate
buffer
pH 7.13.
2,3-DPG
was
assayed
according
to Beutler’s
modification
of Krimsky’s
method.14
RESULTS
2,3
and
DPG
Oxygen
Determination
fractions
obtained
by a regular
rather
narrow
Affinity
of
in
decline
range
DIscussIoN
AND
in Red
Blood
Cell
Fractions
2,3-DPG
in
10 individuals
young,
intermediate,
showed
that aging
of 2,3-DPG
of 2,3-DPG
(Fig.
level
1 and
Table
(Table
1).
a
Table
1).
regular
The strict
intermediate
#{176}Ourresults
difference
of
significant.
decrease
parallelism
and old
red
of
P50
between
blood
are expressed
as tmoles
2,3-DPG
between
“old”
during
the
process
category
average
to
of 60%
the
young
fractions
aging
of 2,3-DPG
in Fig.
and
1.
seen
2,3-DPG
per
and
“young”
g Hb.
cells
If expressed
would
be
fell
in a
decrease
fraction.#{176}
showed
of
the evolution
cells is clearly
Age
and
old red blood
cell
of the cell is followed
1). Each
An
of 2,3-DPG
was found
in the old cells
as compared
The oxygen
affinity
of the different
red blood cell
tively
of Different
(Fig.
correla1 and
P50 in young,
per ml
lessened,
RBC
but
the
still
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
2,3-DPG
CONTENT,
These
OXYGEN
data
confirm
affinity,8
and
Bunn
2,3-DPG
content
experiments.
of
method.
In
2,3-DPG
content
of
effect
were
exposed
cells
not
HjelmOL
of the
not
to
2,3-DPG
others
in
of
cubation
with
tent.9’15
In
depleted
vivo
We
in
vivo
fractioned
total
was
depleted
the
was
by
source
nieasured
in aliquots,
containing
0.01
of
fraction
and
regain
the
obtained
in the
lost
along
be
youngest
to
total
samples
some
“total”
red
procedure
with
could
for
due
in
stepwise
in
values
of the
errors
In Vivo
usual
was
Aged
the
of
reticulo-
considered
Red
conditions
It
has
most
pure
also
Blood
shown
that
original
observed
CelLi
at 4#{176}C
) the
(ACD
been
their
of
1.-2,3-DPG
drawn
mixtures
into
for
13
and
before
after
in-
2,3-DPG
after
in
Fig.
and
/g
capacity
from
two
young
con-
reinfusion
and
fractions
of
incubation
old
the
for
7.4,
pH
2 it is obvious
Affinity
red
red
that
of RBC
Hb)
1 hr
at
(mm
without
any
content
was
of
saline
2,3-DPG
of Different
Hg
A
inosine.
decline
Fractions
Po
cells.
2,3-DPG
37#{176}C in
15 mM
the
of
was
blood
methods.
2,3-DPG
and
cells
individuals
at 4#{176}C
in saline
after
Oxygen
stored
under
At intervals,
buffer
of
normal
described
days.
phosphate
shown
(mo1es
as
different
the
Content
PG
regenerating
blood
prepared
energy
M sodium
results
2,3-D
( old )
in the
2,3-DPG
also
storing
exogenous
Table
were
2,3-DPG
Fresh
phthalate
fraction
the
values
were
cells
difference
the
including
that
rapidly.9
cells
of
age.
with
sham
no
cells.16’17
compared
have
different
From
the
repletion
stored
blood
declines
inosine
found
the
oxygen
of the phthalate
ultracentrifugation
Unexpectedly,
nearer
on
homogenous.
of
2,3-DPG
method
results
of In Vitro
storage
cell
more
efficiency
to the
fractions
same
Rigas
the difference
of
pronounced
in our
Experimental
all
densest
less
was
cells.
are
youngest
Capacity
vitro
red
latter
red
It is possible
the
being
Restoring
During
level
The
the
only
the
since
phthalate.
Therefore
enough,
the
and
However,
cells
fraction.
likely
used,
red
samples
to phthalate.
we
old
“old”
total
are
Edwards
the greater
as compared
using
intermediate
by
to
and
the
exposed
centrifugation
cytes.
and
separation
of “young”
2,3-DPG
instead
phthalate
cell
AGE
made
due
465
CELL
on 2,3-DPG.9
young
probably
contrast,
P50 and
cells
is
BED
observations
co-workers
between
method
esters
the
the
and
This
AND
AFFINITY,
Age
02)
Subjects
Examined
Total
Young
Intermediate
Old
Total
Young
1
2
3
4
5
6
13.1
15.8
16.3
15.2
16.3
18.2
18.1
17.8
18.0
16.7
16.0
11.8
11.4
12.6
11.5
11.1
11.9
6.7
6.8
7.6
7.7
7.4
6.8
8.1
28.0
30.5
30.0
29.5
29.0
29.5
29.5
30.5
32.0
32.0
31.5
31.0
32.0
30.5
Intermediate
27.5
27.5
2.8.5
27.5
27.5
27.0
Old
8
14.0
16.8
11.4
6.3
29.0
32.0
27.5
9
10
14.2
15.2
16.7
16.9
12.9
13.0
9.0
6.5
29.0
29.5
30.5
30.5
28.0
28.0
24.5
24.5
24.5
27.0
25.5
24.5
25.5
25.0
26.0
24.0
Mean
14.7
± 0.9
17.2
± 0.7
12.0
± 0.5
7.3
± 0.7
29.3
± 0.4
31.2
± 0.5
27.7
± 0.2
25.1
± 0.8
14.7
7
14.2
14.3
± SD
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
466
HAmA5,
r
Day
r5
st
LABIE,
oF
AND
KAPLAN
storage
th
11.4
9.5
CASE
1
8.2
6.665
J31
65
I
0’
0
a.
0
T
V
0
1.6
T
V
0
0
before
nosine
17.5
18
16.9
oiler
nosne
15
E
CASE
2
10’
S.
14.7
FL
0J
Fig.
2.-Capacity
irk the
is more
of inosine
In
in
completely
contrast,
young
throughout
2 wk
compares
the
with
old
of
same
range
the
These
cells
findings
cells
is
blood
cells.
The
energy
rich
rather
to
the
capacity
The
question
is located
between
on
before
in
the
vitro
impairment
of
still
remains
the
pathway
triose
phosphates
to
and
and
2,3-DPG
on
in text).
that
their
2,3
these
DPG
cells
content.
restoring
the
2,3-DPG
capacity
striking
if
13th
day
have
a 2,3-DPG
merely
vivo
synthesis
in
reflect
to
that
one
ohlevel
the
stored
red
aged
red
depletion
senescent
unaltered
metabolic
handicap
to the
the
in
vivo
would
remains
inosine
in
phenomenon
in
2,3-DPG
the
observed
observed
might
in
from
day
2,3-DPG
details
inosine.
of
whether
to restore
(see
fractions
that
the
2,3-DPG
leading
both
from
2,3-DPG
cells
is particularly
with
to bind
as
their
decrease
whereas
of
13th
cells
since
different
loss
of
red
replenish
young
day,
the
of hemoglobin
on
most
incubation
that
the
to
(0)
storage
phenomenon
in
5th
the
intrinsically
compounds,
on
cells
capacity
This
old
preliminary
older
00.5
1
(Y) and
their
retain
6.1
V
after
the
observed
suggest
blood
young
and
lost
storage.
increment
tamed
(T),
cells
of
E2
0
1
before
pronounced
almost
2E2
8
3.4
of total
presence
have
or
8 14.7
V
0
T
V
main
of
be
due
cells,
since
in old
cells.18
in
glycolytic
these
cells
route,
2,3-DPG.
REFERENCES
1. Chanutin,
A., and
Curnish,
R. R.:
Effect
of organic
and inorganic
phosphates
on the oxygen
equilibrium
of human
erythro-
cytes.
Arch.
Biochem.
121:96,
1967.
2. Benesch,
R., and Benesch,
R. E.: The
effect of organic
phosphates
from the human
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2,3-DPG
CONTENT,
erythrocyte
hemoglobin.
mun.
OXYGEN
AFFINITY,
AND
on the allosteric
properties
of
Biochem.
Biophys.
Res. Corn-
26:162,
1967.
Enoki,
Y.: The interaction
of hemoglobin
and
its subunits
with
2,3
diphosphoglycerate
Proc.
Nat. Acad.
Sci.
U.S.A. 61:1102,
1968.
4. -,
and
-:
Intracellular
organic
phosphates
as
regulator
of oxygen
release
by
3.
-‘
and
-,
haemoglobin.
Nature
(London)
221:618,
1969.
5. Beutler,
E.: “A shift to the left” or “a
shift
to the
right”
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regulation
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Blood 33:496,
1969.
6. Bunn,
H. F., and Jandl,
J. H.: Control
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New
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282:1414,
1970.
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D. J., and
Kennedy,
A.
C.:
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1954.
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M. J., and
Rigas,
D.
A.:
Electrolyte
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46:1579,
1967.
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H. F., May,
M. H., Kocholaty,
W.
F., and
Shields,
C. E.:
Hemoglobin
function
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9a. Hjelm,
phoglycerate
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healthy
types
M.: The content
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some
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erythrocytes
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1969.
10. Danon,
D., and Marikovsky,
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Y.: Deterof red cell
RED
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467
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11. Brok,
F., Ramot,
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Danon,
D.:
Enzyme
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Israel
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G., and Benesch, R. E.: Determination
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A. J., and Huehns,
E. R.:
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14. Beutler,
E., Meul,
A., and
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L. A.: Depletion
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acid in stored
blood
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15. Oski, F. A., Travis,
S. F., Miller, L. D.,
Delivoria-Papadopoulos,
M.,
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E.:
The
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L.: The
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1969.
17. Valeri,
C. R., and Hirsch,
N. H.: Restoration
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adenosine
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1969.
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S., and
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Unpublished
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From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1971 38: 463-467
2,3-Diphosphoglycerate Content and Oxygen Affinity as a Function of Red
Cell Age in Normal Individuals
STAVROS HAIDAS, DOMINIQUE LABIE and JEAN-CLAUDE KAPLAN
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