Download Red Cell Aging. I. Surface Charge Density and Sialic

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Red Cell Aging.
I. Surface
Charge
Density
Sialic
A cid Content
of Density-fractionated
Human
Erythrocytes
By G.
It has
red
been
cell
V.
F. Seaman,
suggested
that
surface-charge
R. J. Knox,
decreases
density
F. J. Nordt,
in
ORMAL
120 days
cells are
primarily
istics
HUMAN
and the
RED
degree
CELLS
circulate
of random
cell
eliminated
by phagocytic
in the spleen,
but also
of the
old
red
cell
cells
in the
which
lead
D. H.
Regan
charge
density
for the extreme
5% fractions based on the electrophoretic
behavior
of the cells. However,
slightly
lower levels
of sialic acid were
consistently
observed
in the densest
red cell subpopulations.
These observations
and other
cited
evidence are consistent
with
the view
that
during its life span in vivo, portions
of the
red cell membrane
are lost as the result of
innumerable
cell-cell
and cell-vessel
wall
contacts
during
the passage
of the cells
through
the circulatory
system.
Such losses
would account for decreases
in the level of
membrane
constituents
per cell, such as
sialic acid, but would
not require
that the
concentration
of those constituents
be altered in the remaining
membrane.
Thus
such features
as surface-charge
density
could remain
unchanged
even though
the
total sialic acid content
per cell was reduced.
accompany
aging
in vivo and
reflect
alterations
in
the cell surface
which
play a critical
role
in the recognition
and elimination
of effete
erythrocytes
by macrophages
in the reticuloendothelial
system.
The bases
for this
suggestion
are reports
that
the surfacecharge density
progressively
decreases
for
red cell subpopulations
sampled
from regions
of increasing
density
within
the
whole
population
where
the least dense
fractions
are enriched
in young
erythrocytes and the most dense in old erythrocytes. We have
attempted
to reproduce
these results and have examined
the relationship
between
cell surface
sialic acid
and cell surface-charge
density
for the extreme density fractions
of fresh human
red
cells. Contrary
to the earlier
reports,
we
observed
no differences
in net surface-
N
and
and
in the bloodstream
destruction
is low.’
of the reticuloendothelial
liver and bone
marrow.2
to its disposal
are
not
for 110Senescent
system
(RES)
The character-
understood
but
are
thought
to involve
changes
in one or more
physicochemical
properties
of the
cell. Both
deformability3
and the net surface
charge4
of the cells are properties
postulated
as possible
initial
key factors
in the recognition
and elimination
process.
would
Progressive
decreases
in cellular
deformability
during
impair
or slow its passage
through
the microcirculation.
erythrocyte
has
become
microcirculation
hypothesis
does
the recognition
sufficiently
rigid
and
is no
longer
aging
of the cell
Once
the aged
able
to
traverse
the
of the spleen,
sequestration
occurs.
While
the deformability
account
for shortened
red cell survival
in some
disease
states,
and eventual
phagocytosis
of the normally
aging
cells remains
unexplained.
From
the
Department
Submitted
April
Supported
Address
Health
©
Blood,
by USPHS
for
reprint
Sciences
/977
of Neurology.
4, /977:
by Grune
accepted
Grant
requests:
Center.
3/8/
& Stratton.
Vol. 50, No. 6 (December),
HL
G.
University
July
/8284
1977
iSSN
of Oregon
Health
National
Heart.
Sciences
Center.
Portland.
Ore.
/977.
from
the
V. F. Seaman,
5. W. Sam
inc.
/2,
Jackson
Department
Park
Road,
Lung
of
Portland,
and
Neurology.
Ore.
Blood
institute.
University
of
Oregon
9720/.
0006-497/.
1001
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1002SEAMAN
El AL.
Another
hypothesis
charge
density
the altered
progressively
basis
that
densities.5’6
have
cells
implied
is due
groups
to
contribute
and
specific
the
The
by
sialic
acid
by the
whose
of the
red
then
its
poly-
increasing
the rapid
on
upon
partial
leads
perhaps
red
carboxyl
cell.9’0
been
receiving
and Morell”
and
de-
to
sufficient
recognition
sialic acid
life span
of
the
by autologous
to the
report
macrophages
normal
we present
a small
Erythrocyte
their
loss
of
phagocytosis
from
mammalian
the cells in vivo’2
in vitro.’5
recognition
and
The
elimination
erythI and
significance
mechanisms
in
either
consistently
was
drawn
citrate
within
method
16 x
but
blood
trisodium
tionated
studies
of the
relationships
between
sialic acid level in density-fractionated
between
the mobility
of “young”
lower
and
level
of sialic
MATERIALS
AND
METHODS
venipuncture
from
healthy
the
electropho-
human
red cells.
We
“old”
red cells,
but do
acid
in old
cells.
Fractionation
Human
a period
100 mm
disodium
of 4-6
Cell
and
Analytical
suspended
Particle
Suspending
NaCI-0.22
media
gradients
volts/cm
Neuraminidase
Vibrio
cholerae
M
for
different
ionic
strengths
the pH
adjusted
to 7.2
were
measured
for
1 hr
NaCI,
or
the
at
27,000
approximately
were
washed
and
high-speed
M
listed
g at
the
three
0.01
the analyses
with
water
currents
yr)
into
It was
frac-
centrifuga-
top
times
and
-30C
in
and
5,,
bottom
in 20-30
potassium
spun
a Sor-
volumes
phosphate
of
buffer,
below.
0.2
used,
by
namely,
NaHCO3
chamber
M NaCI,
and in
in the lower
ionic
bath
at 25.0
±
0.15
M
NaCI
of equivalent
apparatus
and
0.03
ionic
equipped
M
strength.
with
Ag
a cylindrical
chamber
equipped
strength
media.
Chambers
were
0.lC
and
were
operated
at
voltage
<3 mA.
of Cells and Sialic A cid A ssay
N-acetylneuraminic
neuraminidase
were
±
in a cylindrical
suspended
in 0.15
for cells suspended
Treatment
Membrane-bound
medium
25 44
rophoresis
in a constant-temperature
of 5
0.144
(aged
1.5 mg Na2EDTA.2H2O/ml,
to
red cells
with
mobilities
thermostated
(PBS),
donors
method6”6
with
aliquots)
of two
electrodes18
for cells
platinum
electrodes19
ester
corresponding
the
in this
Elect
M sorbitol
Electrophoretic
fractions
fractionation,
saline
phthabate
(Il-mI
adult
tetraacetate(Na2EDTA.2H,O).
anticoagulated
tubes
7.4 phosphate-buffered
286 mOsm/kg,
the
blood
polypropylene
Following
ethylenediamine
hr by
using
centrifuge.’7
collected.
by
or
of Murphy,’7
RC-2B
with
and
of glycoproteins
to
of
the
of senescent
glycoproteins
cells;
leads
particles7
acid,
charge
plasma
removal
of membrane-bound
neuraminidase
decreases
mobility
and
no differences
observe
AgCI
with
surface
observation
separated
on
density
survival
has
of Ashwell
recognize
(age)-fractionated
iron
sialic
if desialylation
erythrocyte
observations
In this
pH
circulating
the
surfaceto
is unclear.
retic
find
were
net
reticuloendothelial
phagocytosis
of these
vaIl
decreased
tuned
of density
colloidal
membrane-bound
holds
aged
are
surface-charge
of the
of
have
RES
RES.
increases
in
of
majority
hepatic
in the
Enzymatic
rocytes
with
vivo
with
acid in erythrocyte
in part by the work
hypothesis
cells
the
interaction
cells
diminished
loss
elimination
sialylation.
removal
the
of
on the
red
a partial
senescent
hypothesis
is founded
on
charge
in the old erythrocytes
Studies
that
The role of sialic
attention,
stimulated
the
macrophages
neuraminidase-treated
lysine8
tion
that
the
surface
charge.
The
decreasing
surface
of their
and/or
provides
and
acid
(VCN,
(NANA)
was
N-acetylneuraminate
released
from
the
glycohydrolase,
cells
by
EC
treatment
3.2.1.18).
From www.bloodjournal.org by guest on August 9, 2017. For personal use only.
RED
CELL
AGING
Typically,
-
2
Behringwerke
I0
x
cells/mi
suspension
pared
-20
37’C.
and
and
and
was
NANA,
A grade,
calculated
U/mI
were
acid
was
in 0.05
were
(TBA),2#{176}alkaline
Total
NANA
released
Total
number
of RBC
f
the
is
packing
suspension,
the final
included
volume
extraction.
a final
fluid
saturated
the
was
pre-
cell count
NaCl-0.0I
acid
cell
I hr at
tempera-
was
assayed
methods.
per
M
for
g at room
Sialic
released
Standard
by
VCN
was
then
fluid
Assay
extracted
used
prepared
by
of the
hypotonic
cells
l0
liters/mole-cm
volume
by
w/v
packed
the
VE
the
in the
with
TCA.
times
of
at each
the
cell
volume
volume
of
of
VCN
the
red
added,
and
assay
a 70%
The
mixture
decantation
of
at room
fluid
NANA
solution
was
cooled
by
ether
of
in
water
in
TCA
an
ice
fluid.
2 volumes
which
the
digests
VCN
followed
supernatant
with
from
in
(TCA)
w/v
the
temperature
supernatant
of
acid
of
final
bath
for
The
super-
diethyl
dilution
ether
factors
step.
in
of
were
NADH.
One
the
2 oxoglutarate
age
of cells
in PBS
by
in
the
per
Henry
cell
at
of
NADH
x
diluted
Red
GOT
cell
lysates
in approximately
to
Units
25’C;
l0
fractions.22
et al.23
centrifugation
mm
to 4.82
aminotransferase,
red
International
oxidation
corresponds
of
by
followed
substrate
for
IU
vivo
expressed
of
nm
340
in
as modified
buffer,23
I ,zmole
at
L-aspartate:
the
volume
assay
Activities
absorbance
of
of Karmen
of a known
phosphate
for
(GOT,
marker
method
sonication
conversion
decrease
the
trichloroacetic
mixed
transaminase
remained.
the
the
with
and
three
as an enzyme
measured
umes
catalyzed
Hct
substances
was
9- l0,
weight
recorded
were
red
VE)
+
Methods
was
was
Hct]
V5
suspension,
fluids
centrifugation
cell glutamic-oxaboacetic
activity
be 0.99,
cell
interfering
sample
of
The
_f.
V
supernatant)
to
red
supernatant
by
water.
was
2.6.1.1)
(IU),
they
10 vol-
establish
that
where
were
calculated
to NAD
using
Karmen
units
unit
from
6.2
=
for
no
I
the
x
assay
of 3 ml employed.
Reticubocyte
were measured
computer
Hycel
counts
were
by electronic
Analysis
reagents
Package
and
tubes
was assumed.
All
reagents
tilled
twice
grade
reagent
and
estimated
particle
Red
Data).
according
following
ware
Hemoglobin
to their
stored
were
in
glass
was
assayed
instructions.
at
distributions
solutions
and
new methylene
blue method.24
with
an Electrozone-Celboscope-PDP
centrifugation
cell density
standard
in pyrex
by the
counting
(Particle
standards
in microhematocrit
0.99
the
of
concentration
with
General
intact
final
was
fluid.
presence
followed
computed
Red
the
7.4,
(RBC/mI)V
of stock
supernatant
15 mm,
EC
M
1000
-
supernatant)(V[l
(NANA/mI
assumed
volume
of
The
give
were
pH
incubated
resorcinol21
NANA
containing
of
a known
and
at
(RBC/ml)
ofsupernatant
on
treatment
natant
PBS
buffer-MIS
NANA.
and
Total
with
mixed
10 mm
Ehrbich,20
(NANA/mb
fraction
V the
Experiments
about
was
of liberated
Calbiochem.
in
units/mi
from
where
to
cells
in PBS,
acetate
it
for
analysis
-
vs
sodium
at 37’C;
centrifuged
for
-
cell
of
100 Behringwerke
-
of erythrocytes
M
suspension
from
a suspension
1.5 ml of the suspension
-
VCN
cell
sampled
obtained
to
of
suspension
of
samples
fluids
added
A stock
aliquot
to this
incubation,
thiobarbituric
was
concentration
of 7.2.
An
added
supernatant
the
a pH
0.3 ml ofSOO
5.6,
Following
ture
solution
an enzyme
hematocrit.
was weighed
pH
VCN
to give
volume
at
CaCl2,
by
1003
were
made
up
jugs.
15,000
g
from
The
for
cell
volumes
5 mm.
A
by the phthalate
analytical
water
fitted
concentrations
8/M
Mini-
as cyanmethemoglobin
Packed
assessed
Cell
grade
the
with
were
packing
ester
measured
fraction
materials
specifications
in water
for
type
water.25
RESULTS
The
hematologic
in Table
1 and
are
in Table
given
parameters
those
for
for
the
cell
2. In experiments
the
phthalate
fractions
1 and
obtained
2 the
ester
fractions
by
the
anticoagulant
of
method,16
are
presented
Murphy
method
was
1 volume
disII
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1004
SEAMAN
Table
1.
Hematologic
Characteristics
of Red Cell
Fractions
Obtained
El
AL.
by the
Phtha late Este r Method
Exp.
Population
RBC
No.
Fraction
Density*
Retics.
(%)
MCH
(pg)
1
lop#{243}%
<1.085
2
lop
3
*Densiy
10%
5%
GOT)
(IU/RBC
x lOfl)
5.5
31.5
96
32.8
1.2
34.6
100
34.6
>1.100
0.6
35.3
87
40.6
18.3
<1.085
6.7
37.2
104
35.8
20.8
16.9
-
1.9
35.6
97
36.7
19.8
11.8
20.1
-
-
-
1.2
35.7
86
41.5
19.6
lop7%
<1.081
3.0
33.4
98
34.1
21.2
Whole
-
1.2
30.3
88
34.4
16.9
7.5
31.5
79
39.9
16.8
4.9
Bottom
2%
>1.097
0.6
listed
is that
of the phthalate
ester
N-acetylneuraminic
glutamic-oxaloacetic
w/v
separation
citrate
technique
counts,
at 20’C.
to 9 volumes
whole
tended
and
13.5
acid.
2H2O/ml
.
mixture
6.6
transaminase.
trisodium
Na2EDTA
locyte
(fg/RBC)
>1.103
GOT:
mg
NANAt
(g/dl)
Whole
Bottom#{243}%
tNANA:
of 3.8%
MCHC
-
Whole
Bottom
MCV
(fi)
cells
of
of blood;
blood.
to have
the
The
higher
bottom
MCV,
5%
in experiment
cells
of
the
GOT,
top
sialic
characteristically
3 it was
5%
acid,
had
1 .5
with
either
and
reticu-
lower
MCV,
GOT,
and reticulocyte
counts,
and consistently
lower
sialic acid levels.
Electrophoretic
mobilities
for freshly
drawn
human
erythrocytes
and density
fractions
obtained
by the phthalate
ester method’6
and the method
of Murphy’7
are presented
in Table
than
10% -of the whole
strengths
of 0.15
mean
electrophoretic
any of the fractions
NANA
was
method,2#{176} and
and
0.03 g mole/liter
at 25#{176}C.No
mobilities
at a given
ionic strength
of whole
populations
analyzed.
estimated
in three
ways:
the resorcinol
procedure.2’
experimentally
and
3. The extreme
density
fractions,
population,
were
examined
by
by adding
to PBS
in the
Table 2.
known
absence
of
Hematologic
red
the
which
constituted
electrophoresis
at
significant
differences
in
were observed
between
TBA
assay,2#{176}the alkaline
Ehrlich
The
recovery
of NANA
was tested
amounts
of
cells,
incubating
NANA
to
for
a red
1 hr
cell
at
suspension
37#{176}C,assaying
Characteristics
of Red Cell Fractions Obtained
by the Method of Murphy
Exp.
Population
No.
Fraction
Median
Density
Retics.
(%)
MCH
(pg)
MCV
(fi)
MCHC
(g/dl)
5%
1.095
6.3
31.8
95
33.5
18.4
9.4
1.102
3.8
32.3
92
35.5
17.6
5.2
1.107
1.8
33.0
82
40.2
16.6
4.3
4
lop
Whole
Bottom
5
6
5%
1.095
6.6
27.4
87
31.5
17.8
10.7
1.103
1.2
27.2
80
34.0
16.0
3.9
Bottom
5%
1.104
0.5
27.6
77
35.8
15.9
2.0
1.098
6.3
30.4
89
34.2
17.8
9.7
1.104
2.1
33.1
87
38.0
17.3
6.1
1.107
0.4
33.2
79
42.0
16.2
4.8
lop
5%
Bottom
tGOl:
GOTt
(lU/RBC
x 1011)
90%
5%
Whole
*NANA
NANA*
(fg/RBC)
Middle
lop
5%
N-ocetylneuraminic
glutamic-oxaloacetic
less
ionic
acid.
transaminase.
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RED
CELL
Table
AGING
3.
1005
Electrophoretic
Mobilities
Exp.#{176}
No.
Population
Fraction
1
2
0.15
10%
Whole
lop
3
±
0.10(46)
(30)
-1.72
±
0.11
-1.10
±
0.06
(30)
-1.72
±
0.09(46)
(48)
±
0.05
-1.74
±
0.09(12)
-1.09
±
0.07(60)
-1.71
±
0.09(50)
1.10
0.07(26)
Bottom
7%
-1.11
±
0.05(32)
-1.72
±
0.09(28)
-1.09
±
0.05
(40)
-1.76
±
0.08(42)
-1.09
±
0.07
(20)
-1.74
±
0.06(20)
7%
Bottom2%
-1.12±0.08(20)
Whole
-1.10
±0.04(24)
-1.10
±
0.07
(20)
-1.82
±
0.07
(20)
-1.11
±
0.07
(20)
-1.84
±
0.09
(20)
5%
5%
1.08
0.06(36)
-1.79
±
0.09(32)
±
0.04
-1.77
±
0.10
(20)
±
0.06(28)
-1.79
±
0.08
(20)
±
0.06(28)
-1.78
±
0.07(20)
90%
-1.08
Bottom
5%
-
5%
fractionation
were
coded
by phthalate
so that
1.10
(20)
-
1.09
±
0.07
(40)
-1.74
±
0.10(30)
-
1.05
±
0.08
(20)
-1.72
±
0.09(20)
-
1.07
±
0.08
(20)
-1.70
±
0.09
ester
during
-1.76±0.09(20)
±
Middle
5%
-1.73±0.12(20)
-1.08
-
5%
Bottom
method;
experiments
the collection
4-6,
of mobility
data
fractionation
the
±
for NANA,
sample
and
then
SD; the number
of individual
calculating
the
cells measured
percentage
appears
recovery
from
an unfractionated
VCN-modified
cell suspensions
treatment
did
plus
not
ether
produce
red
cell
population
red cells with
gave 0.1-0.4
extraction
significant
the TBA assay
method
These
various
experiments
of the assay
procedures
fering
observer
was
unaware
of
VCN.
fg/RBC
the
decreases
nor
in parentheses.
from
data.
No substantial
differences
in the amounts
of NANA
served
for any of the three assay
methods.
Limited
hemolysis
not to interfere
in the NANA
assay
system.
Completeness
acid
(20)
by Murphy
identities.
mobility
treating
treated
(56)
±
lop
All samples
-1.77
0.06
-1.70
Whole
of the sample
0.05(10)
±
0.06(30)
lop
1-3,
±
-1.10
±
Whole
*Experiments
M NaCI
-1.09
Bottom
6
0.03
4%
lop
5
lity (iim/sec/V/cm)t
Mobi
M NaCl
Bottom
Whole
4
on the Basis of Density
-1.09
-
3%
lop
iMean
Electrophoretic
.
6%
Bottom
Red Cells Fractionated
Human
Whole
lop
method.
of Fresh
by VCN
the
(-‘
was
determined
Supernatant
fluids
of TBA-positive
supernatant
in either
fluids
the
level
experimental
recovered
were
ob2%) was shown
of removal
of sialic
from
of
NANA
by re-
from
such
rematerial.
TCA
VCN
diges.ts
assayed
by
in the absorbance
ratio (Abs
549 nm/Abs
532 nm).
established
no significant
loss of NANA
during
any
nor any influence
on the assays
of potentially
inter-
substances.
DISCUSSION
The
need
company
for
reliable
A variety
separation
to describe
the
aging
methods
the
biochemical
and
biophysical
changes
that
red
in vivo
has
impetus
to the
of human
that
of techniques
on the basis
separate
cells
red
cell
populations
have been used, the most
of differences
in density.
given
on
the
basis
may
ac-
search
of their
common
involving
centrifugal
Stratification
of erythrocytes
age.
From www.bloodjournal.org by guest on August 9, 2017. For personal use only.
1006
SEAMAN
according
to cell
combined
production
with 59Fe labeling
of the
in rats by transfusion-induced
populations
age
of rat
has
red
been
cells
life
span
major
would
properties
that
during
of
the
last
ever,
in these
levels
tions,
of leukocytes
which
would
enzyme,
few days
activity
with
contributions
to erythrocyte,
and
Sass
as with
and reticulocytes
influence
the
in GOT
ported
property
decreases
according
of
must
be made
Electrophoretic
density.’8
The
implies
that
brane
at the
electrophoretic
charges
per
approximately
sites could
same
the
GOT
The
cells
of
the
and
can
then any
red cell’s
the
same
cell
age
Statements
which
with
for
as
life span.
of
indicator
cell age
of
the
compares
in the
cell
age
enzyme
favorably
low
cell fracbasis
for
known,
nor
the transition
denaturation
due
to
other
of
levels
characteristics
separation
with
of
the
of
methods
re-
any measured
the life of the
of
are
from
of the
depressed
the hem atologic
2 indicate
that
the relationship
near
the end
the
The
is not
with
age. Accordstudy.
Howliterature,
throughout
some
extent.
associated
fragmentation,
Thus
I and
of cell
in this
reported
red
loss
of
occur
fractions
for cells
cell
cell,
caution.
net
number
of negative
surface”
data
for human
cell.34 If the limit
be
age,
cell density
be regarded
mobility
is recognized
as a measure
of net surface-charge
electrophoretic
mobility
for red cells of different
densities
effective
charges
remains
erythrocytes
of sensitivity
5% or better,
then approximately
lost from
an average
red cell
constant.
per
unit
area
that there
are about
of the electrophoretic
106 or
without
of mem-
It is estimated
somewhat
apparent
l0
from
negative
method
fewer
change
is
charge
in the
mobility.
differences
reported
for
distributed
profiles
to
increasing
activity
“electrophoretic
electrophoretic
cells
centrifugation,
the end of
cell age
region
ofthe
studies
were
enzyme
erythrocyte
in the
to
in the literature.
to the processes
however,
to subsehave been
in vivo.
If
et al.22 have examined
different
levels
for various
enzyme
activities
and have
most
of the cofactor,
pyridoxal
phosphate.33
the red cell fractions
given
in Tables
population
to resort
and MCV
of aging
fraction
probably
represent
a considerwould
tend to mask
any changes
in cell
provides
the most
sensitive
were
used
as indicators
experiments,
the relative
reticulocyte
values
between
least
dense
of red cell
to obtain
one
the MCHC
and
MCV
trends
for the different
cell
2) were
in agreement
with
those
previously
reported
concluded
that GOT
ingly,
GOT
activities
decrease
having
during
toward
MCHC
separated
according
to density.’7’3032
of a column
of centrifuged
erythrocytes
the
without
AL.
fractionation
Suppression
enables
in both
MCH
a consequence
of separation
or MCV
correlation
from
the
ultracentrifugal
in vivo.26’27
polycythemia
ages
from
the most
dense
of older
cells, which
occur
In this study
(Tables
I and
MCH
a range
in a poorer
While
cells
young
cells, those
able range
in age
by
cells
Reductions
apparently
as
determinant
changes
of
produce
thereby
resulting
the older
cells.
red
of different
quent
in vitro fractionation.28’29
reported
in these
studies,
MCHC
is the
disproportionate
demonstrated
El
in surface-charge
by
Danon
and
density
Marikovsky5
ceptance
in the hematologic
literature,
plausible
mechanism
for the elimination
In the present
study
red cells from the
between
and
“young”
Yaari6
have
and
found
“old”
wide
red
ac-
particularly
as such
results
suggest
a
of the oldest
cells from
the circulation.
extremes
of the density
distribution
were
From www.bloodjournal.org by guest on August 9, 2017. For personal use only.
RED CELL
AGING
1007
-::
5%
,-)
Fig. 1 . Mean electrophoretic
mobility
of human
red cells as
a function
of density.
‘
Whole
6%
o, Yaari’s
Pop
data;
mean
stippled
boxes, range
of
mobilities
for data
in
Table 1 ; vertical
bars,
±
SD
calculated
from
Yaari’s
data
with
the
midpoint
of each
mobility
interval
and
the
quency
for each interval
a grouped
data
format.
centages
of
the
SD
to percentage
red
Whole
tion.
±
refer
total
fre-
using
Per-
cell
popula-
population
derived
point
from
090
Yaari’s
1.095
1.100
examined.
red cell
The electrophoretic
populations
obtained
summarized
ences
in Table
for
for red
detected
been
1.105
RED CELL
data.
the
3, showed
extreme
red
cells from
with
ease,
replotted
the
as
(solid
density
is a monotonic
no
cell
line)
line).
that
and
of
The
compared
cell
significant
expected
density,
our
1.120
strengths
procedures,
mobility
mobility
distribution
1, in which
with
be seen from
data suggest
1.115
(g/mI)
at two ionic
fractionation
experimentally
of the density
seen from
Fig.
It can
Yaari’s
function
made
density
populations.
extremes
may be
(broken
ionic strength
ity versus
cell
measurements
by the two
1110
DENSITY
data
differ-
differences
should
Yaari’s
own
have
data6
at
our
data
mobilmobility
indicate
electrophoretic
mobility
is essentially
invariant
with age, at least over
tral -95%
of the red cell density
distribution.
The coefficients
of variation
for the sets of data
in Table
3 average
is probable
that
rather
than
The density
any appreciable
distribution
indicate
that
the differences
tected.
The
of
presented
of 12%
±
1.5
this
variation
dispersity
for the
data
the separation
in mobility
data
an average
cells (19.4
much
on the
reported
in Tables
more
fg/RBC
originates
from
of mobilities
cell fractions
basis
of cell density
by Yaari,
if real,
1 and
2 show
that
creases
in sialic
acid
content
resulting
membrane
with aging
would
tion of sialic acid. Consequently,
on the
electrophoretic
charge
groups,
rather
els of neuraminidase
mobility,
than the
employed
not
from
membrane
young
which
total
and
measures
number
the lack
the
and
been
cells
It
2)
that
de-
contained
than
the old
in contrast
no differences
be noted
that
fragmentation
result
in a change
such losses
of sialic
cen-
factors
effective
to have
neuraminidase-susceptible
NANA
per cell
versus
17.2 ± 1.4 fg/RBC),
even though,
to the findings
of Danon
and Marikovsky5
and Yaari,6
trophoretic
mobility
of the cells were observed.
It should
the
the
particle
population.
(Tables
1 and
was
ought
the
that
5.5%.
instrumental
in the
examined
been
have
physiologic
the plot of electrophoretic
that the electrophoretic
whereas
on
in elecany deor loss
of
in the surface
concentraacid would
have no effect
concentration
of surface
of charges
per cell.’8 The high
of significant
additional
release
levof
From www.bloodjournal.org by guest on August 9, 2017. For personal use only.
1008
SEAMAN
Table 4.
Total
Cell
Whole
Bottom
IBA
223
nmole/mI
RBC
6.3
35
IBA
237
nmole/mI
RBC
6.7
13
VCN
Resorcinol
89 g/iO10
Acid
Resorcinol
lop
36
10
IBA-Resorcinol
135 .tg/mI
RBC
12.2
Acid
IBA
140 pg/mI
RBC
12.7
14
-
-
19.2 mg/100
ml RBC
17.4
37
-
-
2.2
x
g/ghost
22
38
39
-
10
14
348
ng/mg
Hb
10.4
ng/mg
Hb
11.6
Acid,
NANAse
IBA
327
ny/mg
Hb
9.8
Acid
IBA
1.72
omoIe/g
Hb
16.0
Acid
IBA
1.89
j.tmole/g
Hb
17.5
10%
Acid
IBA
1.54
tmoIe/g
Hb
14.3
Acid
IBA
20.7
og/10’
RBC
20.7
14%
Acid
TBA
18.9
ug/109
RBC
18.9
309.3,
MCH
N-acetylneuraminic
were
packed
NANA
indicate
susceptible
41
acid.
made
assuming
a NANA
molecular
weight
of
of 30
pg.
and
1.1
x
1010
perfringens
upon
that
retreatment
the enzymatic
NANA
should
cells.’#{176}
a check
of
literature
NANA/cell)
were
the
of
whole
cell populations
release
is essentially
give a reliable
estimate
absolute
values
compiled
(Table
4).
obtained
for
under
these
conditions
complete.
Thus
the VCNof total
sialic
acid content
NANA/RBC,
and converted,
where
possible,
There
was extreme
variability
arisen
in one or
originating
from
a combination
the contribution
fluids;
(2) losses
of sialic
nate interfering
substances;
ing data used in calculating
Schauer
et al.35 comment
neuraminidase-treated
(adsorption
with formic
acid
red
of
three
ways:
of interfering
that
cells
ether
for
extraction
removal
of the
of lipid
When
these
measures
were
up to 200%,
with
indications
based
on aberrant
absorption
gion.
of
problems
evident
supernatant
and
ion
resin
with
interfering
not
in the
from
same
units
the results
have
to elimisupportfluids
exchange
subsequent
substances
taken,
they
of interfering
spectra
in the
published
be-
in NANA
values
in the supernatant
during
purification
procedures
intended
or (3) erroneous
assumptions
or inaccurate
or normalizing
the results.
TBA
assay
system.
sults were elevated
in the TBA
assay
the
the
in
the values
ranged
300%.
blood
cells may
(I) elevation
substances
of sialic
acid to an anion
exchange
acid)
are both
required
to eliminate
a result
values
into
even
reported
for unfractionated
red cell preparations,
where
tween
6.3 and 22 fg NANA/cell,
a variation
of more than
The divergent
results
given
in Table
4 for human
red
As
40
cells.
Clostridium
the
(fg
11.7
386
tConversions
ofthe
As
9
1.17 pg x 102/RBC
IBA
*NANA
RBC/ml
8.9
IBA
14%
Bottom
Acid
RBC
NANAset
10%
Ref.
No.
Acid
NANAse
11%
Bottom
NANA
Acid,
Whole
lop
Reported
Calc.t
NANA
(fg/RBC)
Acid,
20%
lop
NANA
Assay by
Acid
VCN,
Whole
AL.
Levels Reported for Human Red Cells and Subpopulations
Obtained by Density Centrifugation
NANA
NANA*
Release by
Population
El
from
clean-up
elution
in the
report
that
chromophores
510-540-nm
literature,
studies
rere-
From www.bloodjournal.org by guest on August 9, 2017. For personal use only.
RED
CELL
were
AGING
1009
designed
that
would
of our quantitation
independent
NANA
TBA
assays,2#{176}made
it unlikely
procedure
in exactly
suits by the different
the
values
for
establish
the
techniques
for
assay
methods,
that
were
accuracy,
interfering
the same
way.
methods
under
NANA
validity,
erythrocyte
the alkaline
Thus
given
not
and
reproducibility
sialic
acid.
The
use of
Ehrlich,2#{176} resorcinol,2’
substances
would
influence
the agreement
obtained
conditions
is a strong
influenced
significantly
three
and
each
between
indication
by
rethat
interfering
sub-
stances.
Our average
value
from
whole
human
Cohen
also
et al.4#{176}
and
could
find
of 17.7
red cell
±
1.5 fg/RBC
populations
Greenwalt
no
and
indicators
of the chromophores
interfering
substances
for the sialic acid released
by VCN
agrees
well with
those
reported
by
Steane.4’
Like
of interfering
produced
by TCA
in
and
Greenwalt
substances
the TBA
assay.
ether
treatments
ence on the TBA
results.
The assayed
NANA
and resorcinol
assays
were within
20#{176},,
of the
and
Attempts
had no
values
TBA
from
assays.
Canham32
estimated
that the mean
surface
area and
from
the bottom
l0#{176}fraction
(phthalate
ester
method)
tein,
and
lipid
per
cell.
Cohen
et
distribution
of the losses
portionate
of
removal
membranes
major
tions
remaining
are
cells
not
to ions,45
charge
and
mechanical
density
between
by the
old
of decrease
of
occur
in chemmembrane
pro-
absence
of
However,
if the
for
surface
in partitioning
toward
of the
membrane.46
young
cells
been
the
ratios
behavior
antisera,43
membrane
of
alterain two-
binding
permeability
Differences
have
changes
constituents,
without
dispro-
even
Evidence
agglutinability
macrophages,’5
and
of
properties
altered.
properties
the
basis
et al.’
volume
of red cells
were
about
10% less
constituent.
different
differences
systems,42
by autologous
for some of the changes
of cells has been shown
of these
variations
the
of Jancik
major
membrane
fragmentation
membrane
have
substantially
is provided
phase
aqueous
polymer
lectins,44
phagocytosis
cells
the
ratios
of
represent
particular
in old
components
in old
any
al.,4#{176}
on
and
may
any
inilu-
the alkaline
Ehrlich
We therefore
con-
cells from
the top 20#{176}(,
fraction.
The magnitude
is comparable
to the extent
of the changes
that
of the cells, such as decreases
in total
NANA,
in the phospholipid
suggested
that most
we
examination
to extract
significant
dude
that the 2.8-fold
difference
between
our results
and those
and Schauer
et al.35 is not explained
by interfering
substances.
than those
for
these parameters
ical composition
Steane,4’
by spectral
of
in surface-
invoked
to
account
in these properties.
Now that the surface-charge
density
to be invariant
under
the conditions
under
which
many
measurements
have
must be sought.
been
made,
other
explanations
for these
age-related
ACKNOWLEDGMENT
The
authors
thank
C. Tam
blyn
and
B. Voyda
for
painstaking
technical
assistance.
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Red cell agins. I. Surface charge density and sialic acid content of
density-fractionated human erythrocytes
GV Seaman, RJ Knox, FJ Nordt and DH Regan
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