From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
The
Structure
Phospholipid
and
Composition
of
and
Total
Cholesterol
By T. HALLINAN
M
structures,
are
not
of the
including
demonstrable
amounts
of interest.
blood
cell
several
in
give
have
been
of packed
made
on a few
were
no
and
were
estimate
of
shown
samples
the
Similarly
it has
cholesterol
per
volume
again
no estimate
of the amount
In this investigation,
therefore,
cells
the
amounts
and
and
the
in which
way
localization
of lipids
these
change
in normal
Male
white
in
ard1#{176}in
acid
to
as
were
given
to
as
prepared
Suspensions
then
at
1 per
prevent
iron
previously.1
animals
higher
Twice
a method
cells
in
speed
cent
ferrous
washed
In
utilizing
saline
were
(2000-3000
red
some
by
blood
low
as
at.
total
with
the
conin relation
to
and
erythrocytes
cells
in
specific
at
were
gravity
low
N
by
Gm., were
by Pritchhydrochloric
hydrochloride
prepared
from
samples
compared
of
with
anemic
Ferrebee
erythrocytes.
500
and
and
reticulocytes
( approximately
speed
recommended
0.001
phenyihydrazine
concentrated
centrifuged
g),
here
maturation.
sulphate
injecting
experiments,
their
but
arrived
and
strain,
weighing
120-300
hemorrhage
as described
ammonium
or
compared
discussed
during
of an inbred
by repeated
induced
deficiency,’1
described.’
by
of
was
and
METHODS
AND
norvegicu.s)
Anemia
animals
drink,
described
normal
( Rattu,s
rats
experiments.
per
studies
erythrocytes
reticulocytes
in reticubocytes
red
of
in the reticubocytes
contain
more
total
erythrocytes,5’9
are
of
phospholipid
of cholesterol
in reticubocytes
was
the concentrations
of phospholipid
MATERIALS
used
than
considered
all of these
and
claimed
in rat reticubocytes
are determined
and
of these
lipids
in erythrocytes.
Findings
cholesterol
centrations
more
packed
been
was
components
Reticulocytes
However,
of phospholipid
that reticubocytes
which
investigation
cells
important
investigation.
of reticubocytes
quantity
of
two
to contain
of mixtures
themselves.
unit
both
this
erythrocytes.28
absolute
reticulum,
a quantitative
in the
previously
than
II.
intracellular
endoplasmic
components
cholesterol,
chosen
for
cells
membranous
an
Therefore,
membrane
Phospholipid
membranes,
volume
and
erythrocytes.
of certain
species
unit
contain
mitochondria
Reticulocytes.
Reticulocytes
in
E. EDEN
AND
RETICULOCYTES
AMMALIAN
Rat
g)
Geiman.12
and
This
concentrates
the low specific
gravity
reticulocytes
in the upper layers of the packed cell
column which were separated off and kept for analysis. Four repetitions of this procedure
led to a 4-6 fold concentration of reticulocytes from normal blood. This method
concentrates leukocytes as well as reticulocytes, so it is important that the former
be removed
from
samples
before
analysis.
This was done
by suspending
portions
of the
“buffy
coat”
formed
or centrifugation
in small
volumes
of saline
with a glass rod and aspirating it off.
This
was
repeated
3-5 times
after
each
centrifugation
(9-15
times
in all), removing
almost all of the leukocytes with a minimum
loss of reticulocytes
which
are concentrated
below
the
“buffy
coat.”
The
hematocrits
of suspensions
of cells
in 0.15 M sodium
chloride
From
the
Submitted
#{176}Forthe
mature
Department
Mar.
sake
30,
of
of Biochemistry,
1962; accepted
brevity,
the
word
for
Univers-ity
publication
“erythrocyte”
of Sydney,
Sydney,
July 4, 1962.
is
used
in
this
Australia.
paper
to
mean
the
red cell.
557
BLOOD,
VOL.
20,
No.
5 (Novnnum),
1962
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558
HALLINAN
were
determined
centrifuged
by
at
seconds,
which
cytes.
The
Hlad
blood
cells
and
port,’5
have
determined
being
measured
to
of
smears
Wright’s
cell
6.35
packing
in
brilliant
the
by
10
of
dyne
erythro-
analyzed,
was
samples
cresyl
recommended
were
x
suspension
reticulocytes
EDEN
suspension
of
maximal
of
with
as
cell
impulse
ensure
stained
stain
of
average
volumes
percentage
in
with
cells
Capillaries
an
is sufficient
in
The
counterstained
Natelson.13
receiving
shown
cells
hematocrit.
was
500-1000
of
minutes,
packed
the
analyzed
methanol
30
et al.’4
of
from
micromethod
g for
volume
calculated
the
3,600
AND
of
blue,
red
fixed
Darmady
and
with
Daven-
counted.
Lipids
were
extracted
as recommended
by Reed
et al.,16 with
1 : 1 chloroform-methanol
from samples
of 30-50
per cent cell suspension
in saline
containing
0.6-2
ml. of packed
cells. These
were extracted
once with 15 volumes
of solvent
and twice
with 5-10
volumes
at room temperature
and the extract
was evaporated
to dryness
in vacuo
on a rotary
film
evaporator
at temperatures
less than 0 C. The dried
residue
of the extract
was extracted
with
three
20
evaporated
ml.
to
solution
was
of
by
all
the
with
the
and
the
solution
filtered
lipids
were
then
dissolved
in
total lipid extract.
The phosphorus
washing
that
chloroform
The
termed
unaffected
dicating
aliquots
dryness.
dilute
phosphorus
calcium
present
chloride
was
lipid
as
20
through
sintered
glass
and
ml. of chloroform
and
content
of this solution
described
by
Folch
et
the
was
al.’7
in-
phosphorus.
Lipid
phosphorus
was estimated
by the method
of Allen’8
and was multiplied
by a
factor
of 25 to convert
it to phospholipid.
Total
cholesterol
( free cholesterol
+ any esterified
cholesterol
present
) was
estimated
by
the
Liebermann-Burchardt
reaction
by
a
modification
of the method
of Nlarinetti
et al.’9 Color was developed
in a solution
of chioroform,
acetic
anhydride
and
36 N sulphuric
acid,
added
to the sample
in that
order,
in the
ratios
of 15: 10: 1 ( v/v ) . Absorbency
was
read
at 640 rnj,
10 minutes
after
the
addition
of
the
sulphuric
RNA
was
acid.
Nucleic
determined
phosphorus
by
acids
were
phosphorus’8
fractionated
by
ribose2’
estimation.
and
the
method
of
DNA
was
Schmidt2#{176}
and
determined
by
estimation.18
RESULTS
Reticubocytes
rats,
constituted
while
in anemic
centration
yielded
anemic
stantiably
for removing
nucleic
acids
ratio
and
freed
19 for
cytes,2’22’23
which
centages
the
range
from
latter
have
5 to
in
table
1 for
experiments
of
cells
cent
ones,
The
efficacy
red
the
were
performed
cells
used
with
procedure
per
compared
with
P/C
ratio
in
3 per
cent
average
six
concentration
figures
samples
of
for
total
in
from
reticulo-
for
reticubo-
red
that
cells
the
or leuko-
reticulocyte
percentage
containing
varying
experiments
11
perand
phenyihydrazine-treated
phosphobipid,
erythrocytes
cent
possibility
normal
total
blood,
21;
ani-
Table
1 also shows
the P/C
ratio
in the lipids
of 12 samples
in reticubocytes.
Figures
for lipids
in reticubocyte-enriched
riched
sub-
1.2,22
and
blood
of this
reported
than
from
were
RNA : DNA
ratio
of
in this way. This
of nucleated
bess
Con-
which
20-87
excludes
normal
cent.
reticulocytes
normal
cholesterol
from
50 per
to
previously
This
animals
were
per
containing
any
ratios
samples
blood
cent
by the large
of cells prepared
numbers
total
Bled
reticulocytes.
90
above.
10.
RNA:DNA
red
7 per
from
than
significant
phospholipid,
of
mals.
The
the
to
of cells
greater
total
remaining
than
cent
samples
for
given
are
per
up
as described
seven
contain
the
since
Figures
12
considerably
analyzed
cytes,
to
of
from
nucleated
cells is demonstrated
from reticubocyte-rich
samples
a figure
samples
up
cent
ranged
containing
of leukocytes
averaged
cytes,
they
samples
animals
per
2-3
animals
of cells
samples
cholesterol
containing
enare
and
less
reticubocytes.
of
phospholipid
in
rat
erythrocytes,
found
in
this
in-
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
RETICULOCYTE
Table
PHOSPHOLIPID
AND
1.-Phospholipid
and
559
CHOLESTEROL
Cholesterol
in
Reticulocytes
and
Erythrocytes
P-Lipid
P-Lipid
(mg./ml.
Exp.
1
Cholesterol
(mg/mi.
P.C.)
P.C.)
Cholesterol
Reticulocyte%
6.7
91
2
6.9
86
3
6.7
81
4
6.3
5
6
7
6.1
6.0
6.1
8
6.1
9
10
11
72
1.6
1.4
3.8
4.3
67
67
1.7
3.7
64
5.7
5.6
5.8
1.5
3.8
1.7
3.4
12
5.4
1.6
3.4
35
13
5.3
2.0
2.7
34
64
58
52
38
14
5.5
1.3
4.3
30
15
5.8
1.8
3.2
29
16
5.5
1.5
3.7
25
17
5.4
18
5.0
22
19
5.3
21
20
5.0
21
5.0
22
2.8
± 0J5
1.8 ± 0.20
3.6 ± 0.4
2.6 ± 0.23
1.6
Eryth.
mean#{176}
Figures
4.5 ± 0.17
the
represent
reticulocytes;
11
and
animals.
*The
Where
erythrocyte
vestigation,
concentration
21
are
from
of
bled
applicable,
figures
are
well
agrees
18
7
Nlean
treated
1.8
lipids
standard
averages
with
that
in
samples
the
animals,
deviations
for
six
red
cells
from
enriched
in
phenylhydrazine-
reported.
of
by
blood
are
are
samples
reported
of
remainder
<3
cells
from
normal
Dziemian.24
In
blood.
agreement
with
abservations
on other
mammals,28
it was found
that
populations
of rat red
blood
cells,
enriched
in reticubocytes,
contain
a higher
concentration
of phospholipid
than
do erythrocytes.
Further,
a highly
significant
correlation
exists
behveen
the
content.
between
(r = 0.93; P < 0.001).
However,
the concentration
of cholesterol
reticulocytes
percentage
Since
it is not
extrapolation
indicates
Ce
=
=
=
to obtain
data on mixed
extrapolation
concentration
concentration
concentration
and
reticubocytes;
a population
no
in
while
wholly
and
significant
populations
the
its
difference
of cells
cholesterol
phospholipid
is observed
enriched
in
concentration
and
uncorrelated.
of Phospholipid
populations
of phospholipid
from
how this
Cr
in
Concentration
possible
concentration
Y
are
of the
reticubocytes
in erythrocytes,
percentage
Computation
of the
that
and
reticubocyte
of
in Reticul.octjtes
of pure
in reticubocytes
populations
was performed,
of phospholipid
of phospholipid
of phospholipid
reticubocytes,
can
only
an estimate
be
obtained
(table
1). The discussion
using
the following
below
symbols:
in erythrocytes;
in reticulocytes;
in mixed
populations
of
erythrocytes
by
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
560
HALLINAN
AND
EDEN
U
#{149}
E
65
0
#{149}
#{149}
#{149}
‘0
0.
#{149}
-Phenyihydrazine
treatment
0
‘C
0.
o-
Hemorrhage
1)
0
0
20
40
Fig.
1.-Curves
of
best
fit
relating
too
80
60
Reticulocyte
%
phospholipid
concentration
to
reticulocyte
percentage.
b
=
ratio
=
erythrocytes;
reticubocyte
X
For
any
of
the
mean
cell
volume
of
(MCV)
reticubocytes
that
to
of
percentage.
population
of erythrocytes
and
reticubocytes,
(1)
(100X)Ce+bXCr
(100-X)
In this
the
equation,
value
reticulocytes
1 the
values
the
for
values
b for
the
rat
For
b
1, the
=
For
b
2,
=
X
X and
Y can
is not
of b varies
from
determined
be taken
from
known,
but
data
other
in table
of X and
Y have
1;
mammalian
Consequently,
1.75.2.142527.
values
the
for
in figure
been
plotted
using
2.
line
Y = 0.0195
for
reticubocytes
value
experimentally
of b of 1 and
bX
+
+
the
line
8.84
X
of best
fit is given
by
the
equation
4.85
(2)
of best
+
fit is given
by
the
hyperbola
448
Y=
(3)
x+100
2 and
Equations
Equation
for
Cr,
spectively.
2 gives
while
3 are
values
equation
Thus
equation
represented
of 4.85
3 gives
in figure
mg.
values
3 gives
1 by curves
phospholipid/ml.
of
a value
4.44
for
A and
for
mg./ml.
C
which
Ce
and
B respectively.
and
6.66
is closer
6.8
mg./ml.
mg./ml.
re-
to
ex-
the
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
RETICULOCYTE
Table
PHOSPHOLIPID
AND
2.-Comparison
Normal
(mg/mi.
CHOLESTEROL
of the Concentration
from Anemic
and
Rats
of P.C.)
4.5
Rate
of P.C.)
concentration
In order
to test
the
This
gave
values
the
quently,
the
combined
two
It has
been
in
those
0.82
of
a mean
values
±0.2
Cr
do
obtained
for
not
differ
of 6.7
the
equations
obtained
for
equations
by summating
fitted
curves.
2 and
each
3,
curve.
significantly,
0.2 mg.
±
namely
from
of error
was computed
values
of Y from
was
estimate
in erythrocytes,
of Cr
respectively
of
frequently
nor
content
and
from
Conse-
have
phospholipid
been
per
ml.
of
lipid
in
concentrations
short-term
on
ml.
the
There
that
from
normal
rats
of rat
difference
between
from
anemic
treatment
has
so the
of normal
animals
of phosphodifferent
from
reticubocytes,
content
anemic
is no
or
phospholipid
concentration
samples
been
whether
the
from
the
from
neither
establish
affects
reticubocytes
2 shows
phospholipid
is no significant
cent
reticulocytes
erythrocytes
and
agreement
with
It
in samples
reported
their
suggests
in
reticubocytes
and
as
samples
strongly
the
animals.
no
detectible
value
of 6.7
mg.
reticulocytes.
contained
that
lower
suggest,
of cholesterol
to be present
in red
total
less
esters,
blood
do
cholesterol
and Ponder,5
reported
cells
free
which
of
cholesterol
to dat,32
completely
differ
not
cholesto 67 per
content
uncorrelated.
significantly
from
they
contain.
This
is not in
both
of whom
claimed
that
cholesterol
human
cholesterol
than
cholesterol,
have
are
that
free
of total
containing
up
the cholesterol
not
of total
concentration
Munn31
more
but
concentration
percentage
reticubocytes
concentration
contain
Ponder5
rat
a higher
is noteworthy
If reticulocytes
in
reticubocyte
that
the
of red blood
cells
table
1. In addition,
the amount
of total
reports
of Bodansky9
a significantly
amounts
to
differ
has
Cholesterol
in erythrocytes
of these
made
or phenylhydrazine
content
this
treatment
corresponding
bleeding
as the
terol
cytes.
for
phospholipid
is taken
Retictilocyte
This
of reticubocytes
in experimental
might
to date
were
comparing
Table
calculated
Therefore,
effect
ones.
samples
two
by
normal
produced
though
efforts
of Reticulocytes
hemorrhage29
or phenylhydrazine
reticubocytes,
from
Content
reticubocytes
even
circulation,
Therefore,
bleeding
Phospholipid
that
drugs5’283#{176} or
in the
disproven.
of rat
those
on the
suggested
to
found
short-term
with
Anemia
response
normally
proven
and
estimates
of Experimental
anemia,
tain
and
12
9
cells.
Influence
per
1.06
deviation
to provide
packed
not
of
standard
%
Retic.
of phospholipid
whether
2 and 3 differed
significantly,
an estimate
the squares
of deviations
of the experimental
which
in Reticulocytes
5.1
5.0
determined
mg./ml.
of Phospholipid
Normal
Rats
Anemic
(mg./ml.
5.1
5.0
perimentally
561
they
been
than
than
erythrocytes,
must
conclusively
erythro-
reticulocytes
contain
con-
erythrocytes.
as Bodansky9
substantial
demonstrated
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562
HALLINAN
Phospholipid:Cholesterol
The increase
cells enriched
cholesterol
1 have
0.001
)
and
the
mean
ratio
of
for
erythrocytes
This
enriched
than
per
ml.
is 62
(t
per
P/C
per
in reticulocytes
of packed
the
of red blood
in the total
erythrocytes
of phospholipid
cholesterol,
4.2.
in samples
an increase
by
samples
ratio
mg.
of 1.6 mg.
total
be
P/C
6.7
figure
would
the
greater
contain
concentration
reticulocytes
of phospholipid
accompanied
Therefore,
a significantly
taking
their
in the concentration
in reticulocytes
is not
Reticulocytes
.
EDEN
Ratios
concentration.
table
AND
cells
ratio
cent
ml.
of
greater
6.1;
=
P <
of packed
cells
from
1 as
table
a pure
than
in
sample
the
of
mean
P/C
2.6.
of
DiscussioN
The
rat
reticulocytes
investigated
here
contain
6.7
ml. of packed
cells, 49 per cent more phospholipid
locytes
at all stages
of maturity
were
present
analyzed,
of
a
so this
figure
heterogeneous
in reticulocytes
cytes,
than
than
so
appears
that
those
the
regarded
of
a later
at
into
early
Since
which
average
as the
stage
of
phospholipid
cells
mature,
content
of
mature
more
more
maturation
must
ATP
to erythro-
contain
contain
Nothing
definite
it may
be caused
by
per
Reticusamples
concentration
maturity
reticulocytes
they
of phospholipid
rat erythrocytes.
red blood
cell
The
gradually
an
stage.27
as the
reticubocytes.
to decrease
loss of phospholipid.
of this loss but
by
be
reticubocytes
at
erythrocytes
a net
mechanism
must
population
mg.
than
in the
ATP
phospholipid
be accompanied
is known
of
activities
within
the kinetics
the immature
or
cells themselves.
Alternatively
it may
occur
as a result
of the action
of their
extracellubar
environment
upon
the immature
cells.
Crosby
invoked
a mechanism of the latter
type when
he postulated
that the decrease
in the concentration of lipids
in reticulocytes
hand,
however,
the decrease
cytes
pases,
during
similar
cellular
maturation
to the
components
It has
been
over
of
concentration
might
the
of
acids,35
two.
ATP2T
which
that
reticulocytes
reticulocytes
from
from
animals
normal
Reticubocytes
and
are
is evidence
from
able
that
to
they
evidence
of studies
et al.37
the
that
most
of
However,
lipid
in
Dziemian’s
the
reticubocytes
assumption
cell membrane
also.3
the high
concentration
to the
increased
quantity
the
no
lipid
previous
On
the other
in reticulo-
phospholidegrade
other
of
have
anemic
anemic
not
erythrocyte
Ponder
worked
on this
of lipid
in reticubocyte-rich
with
no
animals
suffered
with
the
contain
high
from
serious
free
functional
of
with
the
the
cell
localization
so no evidence
exists
in this cell is associated
material
con-
abnormal
is lacking.
et al.3#{176}
and Tishkoff
is associated
assumption
red
the
significant
hemoglobin
studies
induced
of
animals
revealed
that they
are
by Erickson
experimental
membrane
animals
comparison
synthesize
have
been
made,
that most of the lipid
of cell
from
However,
alterations.
Definite
experimental
It is generally
agreed
as a result
membrane.
the spleen.33
of phospholipid
to the action
of intracellular
and
proteases34
which
abnormal.
in
reticubocytes
between
concentrations
by
period.
be
phosphobipid
in
difference
this
suggested
anemia
centration
amino
may be due
ribonucleases3#{176}
frequently
experimental
might
be mediated
in the concentration
to
of
support
with
the
when
he attributed
blood
cell populations
contributed
by
the
large
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RETICULOCYTE
PHOSPHOLIPID
reticulocytes.5
er227
more
AND
However,
reticubocytes
than
erythrocytes-hence
but less cell membrane
spherical
erythrocytes.
563
CHOLESTEROL
are
more
spherical29’38
as well
a unit
volume
of them
material
than the same volume
Therefore,
if their
membranes
as
larg-
should
contain
of smaller
and
contain
the
same
not
less
amount
of lipid per unit area
their
lipid is localized
as those
of erythrocytes,
as Ponder
implies,5
and most
of
in the membrane,
reticubocytes
should
contain
less lipid
per
than
shows
ml.
This
lower
of packed
investigation
cells
concentration
lipid
of total
concentration
is 49
concentration
erythrocytes.
that
reticubocytes
cholesterol
of phospholipid
abandoning
Ponder’s
lipid-rich
of
chondria
and
erythrocytes.1
tion
of lipid
thesis
that
with
a large
with
them
and
large
P/C
of erythrocytes.
can
be
lipid
a slightly
their
that
a similar
phosphoThis
accounted
composition
have
P/C
ratio
without
for
the
shown
mammalian
phospholipid
cells
high
a high
mitoin
concentra-
cells.394’
so the
hypo-
of reticubocytes
concentration
compared
Electron
contain
demonstrable
to contain
of the
the
not
cell
that
a subin phospho-
they
are
high
for
membranes.
that
in other
for
in these
cell
which
been
account
ratio
their
show
reticulum,
proportion
would
than
reticulocytes
structures
a substantial
sociated
than
other
sectioned
endoplasmic
These
contain
but
and
erythrocytes,
if it is postulated
phospholipid
of reticulocytes
is localized
structures
fixed,
an
of
indeed
erythrocytes,
in reticulocytes
cholesterol-poor
micrographs
greater
assumption
membranes
of reticulocytes
stantial
proportion
of the
the
cent
per
may
than
with
is as-
of phospholipid
erythrocytes.
SUMMARY
1. The
per
rat
reticubocytes
ml. of packed
2. Reticubocytes
tion
of phosphobipid
concentration
differ
significantly
4. The
icantly
49 per
anemic
contain
from
of total
from
normal
cytes.
This
reticulocytes,
(P
<
finding,
leads
of these
cells
cellular
structures
together
to the
may
be
than
the
1. Le
mg
que
de
phospholipido
erythrocytos
2.
ratto
per
with
4. Le
ab
significativemente
proportion
same
concentra-
examined
reticulocytes
ratio
observations
a substantial
and
does
from
erythro-
on the ultrastructure
proportion
of the
cholesterol-poor,
endoplasmic
in be presente
de
is signif-
in lipid
of
lipid
intra-
reticulum.
INTERLINGUA
examinate
animales
cellulas
anemic
concentration
de phospholipido
como
3. Le concentration
de cholesterol
significativemente
phospholipid
studio
paccate,
i.e.
contineva
49
pro
6,7
cento
±
plus
de ratto.
Reticubocytos
differe
from
phospholipid-rich,
IN
ml
lipid
in
as mitochondria
de
reticubocytos
of
reticulocytes
corresponding
SUMMARIO
0,2
in the
with previous
hypothesis
that
associated
such
mg.
in erythrocytes.
ratio
0.001)
0.2
±
rat erythrocytes.
to contain
the
animals.
cholesterol
that
phospholipid:cholesterol
greater
6.7
cent more
than
animals
appear
as those
3. The
not
examined
cells,
from
ab
ilbo
grande
apparentemente
be
normal.
examinate
mesme
non
in erythrocytos.
phospholipido
plus
contine
reticubocytos
ab animales
total
in le reticubocytos
a cholesterol
(P
<
0,001)
in
que
lipido
be
ab
reticubocytos
correspondente
es
pro-
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564
HALLINAN
portion
in
servationes
que
un
lipido
in re
ab erythrocytos.
be ubtrastructura
proportion
associate
substantial
cholesterol,
i.e.
del
per
exempbo
constatation-insimul
reticubocytos-supporta
lipido
intracellular
structuras
con
Iste
del
in
que
iste
es nc
be mitochondrios
le
es
possibibemente
phosphobipido
e be reticubo
EDEN
previe
obhypothese
con
cellulas
in
AND
e povre
in
endoplasmic.
ACKNOWLEDGMENTS
The authors
wish to thank
Professor
J. L. Still for generously
investigations.
One of us (T. H.)
is also
indebted
to the
Industrial Research
Organization
and the Wool Research
these
and
providing
facilities
Commonwealth
for
Scientific
Committee
for financial
support.
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1962 20: 557-565
The Structure and Composition of Rat Reticulocytes. II. Phospholipid and
Total Cholesterol in Reticulocytes
T. HALLINAN and E. EDEN
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