From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
The
Chemical
Composition
Cells,
including
Variability
By
B
G.
HANS
ECAUSE
water
preliminary
cent
presents
of 22 normal
H.
Human
Centrifuged
JONES
adults
to
the
a study,
results
in the
in
its the mineral
defimsition
of
with
from
state
Blood
Cells
MACLACHLAN
patiemsts
obtaimied
post-absorptive
Red
E.
AND
DATA
show
wide
variation
humami
red cells,’7
further
undertaken,
disturbances.
fhi
communication
samples
Normal
among
BERMAN,
AVAILABLE
contemst
of normal
was
composition
H.
KEITEL,
of
metabolic
analysis
wit-h
and
their
of
iso history
blood
of re-
illness.
M
The direct
by
was used for red cell analysis
method
Crabtree
and
ETHODS
Maizehs,3
Solomon,7
Peters
to inssure
ammd Van
accuracy
Slyke,8
for reasons
and
Ponder.9
indicated
Blood
samples
of 10 to 20 nsl. were drawn,
without
the use of a tourniquet,
from the antecubital
vein into
unoihed
silicone-coated
syringes
which
contained
a trace
of heparin.*
The blood was kept
in the stoppered
syringe
at room temperature
with nmeghigible
loss of CO2. When
CO2 or
pH was to be determined,
a separate
sample
of blood
was kept iced unmder mineral
oil.
A well-mixed
ahiquot
was transferred
immediately
to a rubber-stoppered
Wintrobe
tube
for hematocrit
determination.
An amountof blood,
a
determined
by the hematocrit
or
whole
blood
hemoglobin,
was thens transferred
to a siliconme-coated,
rubber-capped
ButlerCushnsan1#{176} type constricted
censtrifuge
tube,
so that
the buffy layer
would
be located
in
the constricted
and buffy layer.
portion
of the
tube,
pemnsittinng
the
complete
rensioval
of supemnatant
plasma
at 4100 RPM
(mean radius of gyration
of 15
for 45 minmutes at 20 C. Aft-er centrifugation
the supernatant
plasma
and bully
layer
removed.
The entire red cell sample
or desired
portion
thereof
was pipetted
into a
ground-glass-stoppered
weighing
bottle
and weighed.
The sample
was then diluted
an
accurately
measured
red cells.
which
Total
i)y
were spun
volume
of
distilled
water,
of
about
four
ml.
of
water
yielded
phosphorus
a modification
of
good
sensitivity
(from
was
determined
by the
the
Vohhard
method,
0.05
to
0.4
met-hod
the
Pediatrics,
and
Children’s
Electrolyte
Submitted
*
silver
April
nitrate
and
SubbaRow
anmd nitric
acid
heparin,
significantly
accepted
acknowledge
Hospital
50mg.
alter
and
per ml. Trace
the
analytic
Genmerah
Massachusetts,
Heart-
S. Department
1954;
gratefully
General
Massachusetts
Boston,
Nationmal
U.
20,
Service,
School,
Metabolism,
authors
Upjohn
not
Medical
Service,
Massachusetts
did
Medical
Harvard
Public
Health
14, Maryland.
The
were
tamed
with
gram
of
nsmM penS liter).
of Fiske
(12) ; chloride
being
ratehy
as recommended
by Wilson
and Ball;13’
14 magnsesiuns
by a modification
of Briggs
and Dennis;15’
16 calcium
by the method
of Fiske anmd Logan;17
by the method
of Van Slyke and Neihl.’8 The water
content
determined
mens
for 24 hours
in an oven at 105 C.
From
per
cm.)
Thoroughly
mixed
ahiquots
of this solutions
were used for various
analyses.
and potassium
were determined
h)y flame
photometry
using
a Barclay
flame
with an internal
lithium
standard.”
It was found
that hensoghobin
and ferrous
ions did not significantly
affect the sodiuns
and potassium
determinsations
in the
Sodium
photometer
anni ferric
range
The specimens
Institute,
Nationnal
of health,
for
pubhicltt-ions
the
suggest
R.
W.
Junme
Berhinmer,
amounts
result-s.
370
11,
K,
speci-
of
of Kidney
of
Welfare,
of Drs.
Inmstitutes
P and
drying
Health,
Bethesda
1954.
assistance
Nationsal
of Na,
and
sepa-
1)epartment
Laboratory
Insstitutes
Educationm,
ionms and
by
Hospital,
anmd the
added
of thse method
and CO2 content
Cl presenst
A. M.
of
Butler,
healths.
its the heparins
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
KEITEL,
TABLE
1 -Coefficients
Per
Kg.
red
Per
Kg.
plasma
of Variation
of 90 mM
times
AND
per
per
mM
2.-Composition
Kg.
An alyses
on Individu
Potassium
0.18
0.45
3.1
3.0
0.15
1.50
0.65
standard
0.9
deviation
has
Sodium
a one
of Erythrocytes
from
Different
Portions
of
cent of cells by volume*
per cent of cells
Grams
per 100 ml
Hemoglobin,
Grams
per
Potassium,
mM
Contamination
as
Calculations
The
and
analytic
converted
values
to
of solids
Kg.
per
When
data
Kg.
are
of plasma
are
specific
solids
presented
by dividing
to
as
of
per
The
water
=
in water
cells
as fraction
consventional
quantities
per
and
would
content.
increase
if referred
to
solids
as
a reference
water
content
the
46.8
liter,
141
they
may
of plasma
by
cell
be
of cells
by
The
water
the
cells.
by
were
fraction
conversion
is,
as
to
Amounts
the
1,025
from
follows:
converted
of red
dividing
Kg.
values
plasma.
or
gravity
per
former
cells,
specific
of concentration
red
is referred
alone.
will
avoid
An
additional
in cell
Kg.
weight
quantities
per
liter
approximate
example,
per
may
misleading
loss
be
of 53 Gm.
of 5.6 per
the increase
nso change
changes
useful
it’)
(H-’)
as quantities
the
solids,
apparent
><
solids
water,
in potassium
cell water,
to cell
-
of cells.
of cell
For
an
>< (1
quantity
_
of presentation
or volume
mineral
cells;
content
or milhimoles
trapped
quantity
Kg. solids
water
of the
methods
weight
produce
to whole
potassium
Kg.
Kg.
=
quantity
Kg.
W
for
per
per
53.9
Data
dividing
made
approximate
quantity
Kg. cells
2)
as
was
44.6
plasma.
1)
where
by
quantities
to amounts
Cell
as grams
solids
quantities
by 1.1 the
converted
gravity
cell
252
37.4
158
method.
Red
constituents
Kg.
No correction
cell
T1824
of Presenting
per
cells.
Kg.
178
60.1
the
2
0.0
32.6
1873
276
70.2
by
of cell
quantities
per
quantities
Methods
“Bottom”
2
307
solids
determined
of
Cells
0.2
30.2
2007
2146
solids
RBC
-
error
Centrifuged
2
mM
Chloride,
Phosphorus,
per
RBC
Kg.
3.5
“Middle”
6.0
28.3
per Kg. RBC
solids
mM per Kg. RBC solids.
mM
per Kg. RBC solids
Sodium,
*
Kg.
per
Phosphorus
of Erythrocyte
“Top”
Water,
Chloride
cells.)
Type
Plasma,
per
Reticulocytes,
al Samples
Water
red cells
Kg.
371
MAcLACHLAX
Cen t, Of M uiltiple
in Per
of potassium
90, or 0.41
TABLE
JONES
cells
(A value
.0045
BERMAN,
form
in
in
weight
or volume
for
of water
electrolyte
considering
result.
content
osmotic
Thus
due
relations
or
changes
from
cent
if the potassium
would
be 9.0 per cent.
would
of cells
describing
a Kg.
of red
were
referred
However,
the
to
use
changes
is in
if
of cell
in
terms
water.
RESULTS
Table
individual
1 gives
samples.
the
means
of the
coefficients
of variation
of multiple
analyses
of
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
372
CHEMICAL
COMPOSITION
CONCENTRATION
OF
MINERAL
OF
HUMAN
SOLUTES
CELLS
RED
IN CENTRIFUGED
RED
CELLS
300
=
CI
200
iaIP(RK6H10
K
noc
0’
CI
K
MIDDLE
CELLS
BOTTOM
CELLS
--
-
-
Top
CELLS
L
K
p. PHOSPHORUS
1.-Concentration
FIG.
Table
2 amid
figure
red cells taken
middle
port-ion
by
volume,
of
of the
reticulocytes
of “top”
tam
of these
cells and
These
red
more
results
cells,
samples
not
and
contents
less
1 presemst
from
about
of centrifuged
does
tamination
of mineral
data
the
the
cells
substances,
the
three
higher
than
but
more
sodium
than the “middle”
indicate
that when
the
umsequal
sampling
red
the
in
variatioms
cells
composition
portions
indicates
of
the remaining
of 2 per cent,
that
plasma
con-
the differing
composition
of cells. The per cent
sodium,
potassium,
chloride,
and phosphorus
for
water,
are
illustrating
in centrifuged
upper
and lower
6 per cent and
red blood
cells. The plasma
content
from
account
sohut-es
may
the
other
cells.
The
“bottom”
than
the
“middle”
hemoglobin
cells.
direct
method
occur.
Analysis
is used
of the
for
cells
and
the
entire
con“top”
analysis
centrifuged
of
red
cell specimens
from
which
the plasma
and buffy
layers
have
been
carefully
removed
as described
above
avoids
such sampling
errors
as well as those
inherent
in the indirect
method.
The data
presented
subsequently
were
obtained
from
amsalysis
of
of 11
the
entire
males
TABLE
Subject
cemit-rifuged
3 amid 4 list data
amid 11 females.
Tables
3.-Electrolyte
Plasma,
Gm/Kg.
red
obtained
There
cell
specimen.
from
analyses
are no noteworthy
Composition
mM-Kg.
of Normal
of the plasma
sex differences
red
other
blood
cells,
mM/Kg.
solids
solids
H2O
Na
K
Cl
CO
H20
K
Na
Ca
Mg
1.2
15.9
Cl
Ack
915.4
142.4
4.13
100.1
31.8
2008
919.2
144.0
97.8
25.3
1892
Cm
915.0
141.0
98.9
29.7
1974
43.7
264
-H
-
Gg
909.7
143.9
4.18
4.06
4.02
46.3
38.2
277
Ag
97.9
-
142.5
4.05
101.9
260
--153
Gm
916.7
142.2
3.81
103.0
1922
42.2
48.0
39.4
262
917.3
28.5
30.8
26.5
1871
Gi
265
Hy
915.8
141 .3
3.82
101 .3
30.8
2052
40.8
280
-
Ki
918.9
141 .5
4.09
102.6
30.5
1-Ic
917.0
143.2
4.19
32.6
1914
1987
279
274
-
Org
907.8
143.3
3.71
102.5
28.2
1909
51.8
39.7
42.2
271
-
St
912.1
143.3
4.41
98.7
28.7
1927
43.0
263
0.5
10.0
141
Mean.
914.7
142.6
4.04
100.4
29.8
1942
43.2
267.9
1.2
1l.4
151.1
S.d...
3.8
cells
than
Blood-Males
Red
Gm/Kg.
and
1.04
0.20
99.3
2.0
2.33
1910
44.1
3.9
252
8.8
1.5
-
166
P
57.8
160
51.5
-
152
141
59.8
57.4
-
147
60.2
-
167
59.8
57.4
57.9
55.6
57.0
-
10.21
9.4
139
150
-H45
-
55.0
8.9
57.2
7.4
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
KEITEL,
TABLE
BERMAN,
4.-Electrolyte
Plasma,
JONES
Composition
mM/Kg.
of
Gm.,
Gm/Kg.
Subject
AND
Na
K
Cl
CO3
Na
4.20
108.0
4.38
100.2
139.9
Hp
104.9
96.7
Kh
919.2
139.4
Mns
913.3
139.5
Pms
907.8
137.3
Qu
906.9
1:0.-
3.63
4.05
4.03
3.65
4.32
3.86
Sc
Tm
908.1
m19.
3.88
101
917.8
118.9
3-97
!‘l
Wr
911.9
141.1
3.94
102.7
28.4
Mean.
913.0
139.9
4.00
102.0
28.2
1954
0.23
2.9
2.7
140.7
3.6
S.d...
Red
H20
141.1
141.2
Fl
Blood-Females
Kg.
916.2
919.2
912.4
910.5
Br
Normal
blood
cells,
mM
Kg.
solids
solids
HO
Ad
373
MAcLACHLAN
1.2
31.2
26.2
38.2
278
-
164
40.0
299
-
-
159
78.3
2043
1939
37.1
279
-
-
164
58.0
40.0
285
0.6
150
1843
270
-
-
160
54.7
56.0
-
-
149
57.0
151
161
63.5
-
1941
1993
356282
196g.)
3q.5271
1886
28
0
1969
30
3
285
1.
1890
36.1
269
-
35.9
3.7
276
.
4
OF
65
PLASMA
NORMAL
P
1947
103.7
25.0
Cl
2073
103.2
1/1.G
Mg
Ca
38.1
35.3
100.0
RELATIONSHIP
IN
29.7
K
SODIUM
MALES
260
--,
----
260
1
-
12
‘
2
-
1.2
10.7
TO RED
AND
9.4
-
10.8
151
58.5
64.8
147
56.1
136
49.4
154
58.6
CELL
7.0
8.1
-
-
58.4
SODIUM
FEMALES
.
MALES
FEMALES
0
145
.
143’
No
PLASMA
NAV
PER
.
#{149}
#{149}#{149}
-
.
14I
KG
0
0
0
00
I39
RED
of plasma
the higher
sodium
There
is no relation
red cell potassium
The
plasma
(±0.22)
per
than
values
used
for
analysis,
to exceed
about
i
n
34#{149} 40
46
CELL
No
sodium
i
MM
PER
to red
Kg.
concentration
agrees
reported
cell
4.6
normal
mM
KG
SOL
sodium
and
the
I 0 S
in normal
red
red
in
58.
cell
per
plasma
Kg.
cells
4.8
of
per
males
(fig.
state,
data’9’
plasma
values
mM
anmd fensales.
2).
or between
post-absorptive
potassium
(or about
nsales
potassium
with other
recently
obtained
When
properly
obtained*
earlier.
most
i
50#{149}
concentration
in plasma
betweems
the plasma
and
and red cell phosphorus.
potassium
mM
0
0
mO
28
2.-Relationship
#{149}
0
I37
FIG.
..
0
would
the
of 4.02
but is lower
specimens
are
20
miot be
expected
liter).
DISCUSSION
Hlad
cells
*
standing
and
from
Holmes2’
the
amid
blood
Leason
portioms
without
Postabsomptive,
of
upper
samples
and
Reeve22
of compacted
hemolysis,
before
chemical
separation
have
red
cells
recently
contansinmatiomm
of
plasnsa.
demonstrated
contain
more
of
glassware,
that
trapped
plasma
or
excessive
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
374
CHEMICAL
cells
however,
from the lower
the differences
thams
presemit
study
from
COMPOSITION
the
the
upper
comstaminsatioms
portion.
its the
higher
Whets
amouist
potassium
layer
represemsts
alone
would
If centrifugatioms*
the compositiomi
OF
result
caused
of plasma
content
ins cell
reduced
CELLS
centrifugal
force was large,
plasma
were small.
In the
phosphorus
differeisce
in
RED
the relative
of trapped
amid
a true
HUMAN
of
cells
composition
comscemstratiomss
obtaimsed
simsce plasma
of these
a shift
of water
amid/or
electrolytes
from
umicent-rifuged
blood
would
minerals.
from
red cells,
differ
from
that
of
cemitrifuged
blood;
such a differemice
has msot beets observed.
That
miormal
humams
red cells vary
mi compositiois
has been
suggested
previously
: Kurszynski21
noted
differemsces
amonig
umsspums red cells when incimierated
cells
were
which
examined
relate
may
cell
the
its red
to
comitain
imscreased
phosphorus
in
cell
cells.24
by Kay,26
ported
upper
layer
of
phase
potassium
exist
showmi
red
with
red
The
amid
comitrast
loss
“top”
cells
amounts
patients
with
cells.
data,
differences
Ponder’s
population
rabbit
amid
red
water.25
cells
have
An
increase
amsd reticulocytosis
recently
Finch
and
that
potassium
amsemia
has
red
suggest
of cemstrifuged
of
Pritchard27
cemitrifuged
microscope,
to time,
et
reported
has
shown
in
re-
been
reticulocytosis
al.28 have
been
in the
that
the
“top”
cells are
Figure
the only ones which
incorporate
Fe 59.
1 shows
the molal
concentration
of sodium,
potassium,
chloride
amid
phosphorus
in the three
groups
of red cells.
Compared
to the “middle”
and
“bottom”
cells, the younger
“top”
cells have more of these
mineral
solutes.
The
comscemit-rat-ion
of other
substances
known
to l)e presemit
in erythrocytes,
such as
calcium,
comitribute
solutes
magnesium,
sigmsiflcantly
measured
hetweems
cells
glucose
and
to osmotic
should
amid
not
urea
bicarbonate,
pressure
; hemice,
be
interpret-ed
1
Red
.
cells
maimsimig
from
differemst
from
water,
AND
direct
its upper
inherent
3.
layers
the upper
sodium,
method
and
of centrifuged
TJsimig
amid plasma
*
and
inequality
vary
in
richer
phosphorus
composition.
in reticulocytes,
than
the
re-
of red
blood
the
direct
its red
of these
The
relative
2600
on the
of centrifuged
using
cells
a constricted
type
buffy
layer
cells and
differeist
cell popula-
amsd the
cumulative
errors
of the
a constricted
type cemstrifuge
tube,
the means
water
atid mineral
contemstof the erythrocytes
males
and
method
deviations
of 11
layers
cells
sample
from
caused
by
analysis.
normal
found
to have a higher
The sum of the molal
phosphorus
tions
cells
layer,
which
is relatively
potassium,
chloride
and
of amsalysis
lower
its indirect
amid standard
were
4.
osmotic
CONCLUSIONS
censtrifuge
tube to separate
the emitire red cells
from plasma
avoids
the errors
in direct
analysis
tiots
proteinates,
among
those
cells.
The
2.
indicatinig
+
plasma.
SUMMARY
Cells obtaimsed
comitaimi more
as
phosphates
the differences
cells
mimserals
centrifugal
“middle”
amid
11 normal
females
were
is not
in the
force
cells
always
equal
to
the
sum
of the
Males
determimsed.
sodium
cont-emit- of red cells and plasma.
concemitrations
of sodium,
potassium,
chloride
molal
amid
concentra-
plasma.
is 2857
(assuming
on
the
a specific
“bottom”
gravity
cells,
1867
of 1.1 for
ons the
“top”
all
cells).
the
cells,
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KEITEL,
BERMAN,
SUMMARIO
( 1) Erythrocytos
iii
br
reticulocyt-os,
comstine
plus
AND
CONCLUSIONES
ab differemste
Cellulas
al)
composit-iois.
alt-ere
E
JONES
IN
stratos
le strato
aqua,
INTEIILINGUA
del massa
superior,
kalium,
isatriutsi,
375
MAcLACHLAN
de cellulas
cenmtrifugate
que es relativemsiemite
chlorido,
e phosphoro
varia
nc its
que le
cellulas.
(2)
gite
Its le methodo
de
tubo
cellulas
a crusta
msarirnemste
issassa
analyse
imsdirecte.
tTsatste
Esseva
erythrocytos
integre
e et-iarn
directe
le errores
e uts typo
que
le
del
de
a evit-ar
cellulas
typo
cumulative
ab
imsheremste
de
standard
1 1 normal
erythrocyt-os
comistrims-
erythrocytos
le
le errores
causat-e
ordiin le st-rat-os
superior
e
constriisgite
e le deviationes
e le plasma
de
constatat-e
le uso
specimen
le plasma
servi
per differente
le valores
mediams
its le erythrocytos
feminimsas.
de
le
ceistrifugate
le nset-hodo
determinava
e minerales
analyse
separar
e ab
directe
methodo
its le
del
(3)
del
its
phlogistic
inferior
habeva
(4)
directe
cemstrifuge
tubo
in
cemitrifuge
le
nos
del contemsto
de aqua
tssasculos
e 1 1 msornsal
e le
plasnsa
del
mssasculos
phoro
alte contetst-o
de isat-rium.
Le sumsima del concentrationes
niolal
de natrium,
kaliutsi,
chlorido,
e phosin le erythrocytos
tion es semper
identic
con le summa
del concent-rat-iotses
molal
de iste
ums
plus
minerales
in le plasma.
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1955 10: 370-376
The Chemical Composition of Normal Human Red Blood Cells, including
Variability among Centrifuged Cells
HANS G. KEITEL, H. BERMAN, H. JONES and E. MACLACHLAN
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