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Lipid
Content
Acids
and
In Vitro
Into
Lipids
The
By
Lipid
in
MIN0RU
content
and
capacity
14C
into
and
stored
lipids
was
and
lesterol
and
30%
during
in fresh
platelets.
phospholipids
storage
for
glycerides
but
Cho-
mine.
stored
Palmitate
bumin
was
rapidly
energy-independent
free
acid
fatty
there
and
it was
transferred
mechanism
fraction
T
HE
much
and
cells
more
than
role
in
nificant
in
some
for preserving
enzymes.4’5
and
MIN0RU
they
to
but
free
of
has
of erythrocytes,
The
the
Memorial
25, 1971;
Contract
of
signifi-
showed
phospha-
Incorporation
acids
and
into
of
triglyc-
phospha-
reduced.
as
structural
investigated
in
comred
cells
was shown
to play
a sigof red
cell membranes1
Although
appear
its
of linoleate.
into
fatty
LIPIDS
been
cholesterol
stability
of Hematologic
Research,
Providence,
R.I.
12, 1971;
revised February
Atomic
Energy
Commission
that
linoleate
palmitate
was
phospholipids
to
have
of certain
microsomal3
there
is a symmetrical
that
not
and
(3)
into
OF
also
in
produced
incorporation
and
of erythrocytes.2
functions
platelets
palmitate
erides
Specifically,
the structural
platelets
phosphatidylethanola-
of
tidylcholine
It
stored
palmitate
changes:
(1) Incorporation
into
total
lipids
was
linoleate
IMPORTANCE
shape
into
linoleate
acids
and
the
great
and
loss
viability
Hospital,
accepted
March
AT(30-1)3590
particsignificance
mitochondrial
of cholesterol
of platelets,
Pawtucket,
1, 1971.
and NIH
R.I.,
Con-
OKUMA,
M.D.:
Research
Associate,
Division
of Hematologic
Research,
The
Hospital,
Pawtucket,
R. I. Mssiui)
SrEncsR,
M.D.,
PH.D.:
Associate
Director,
of Hematologic
Research,
The Memorial
Hospital,
Pawtucket,
R. I.; and Assistant
of Medical
Science,
Brown
University,
Providence,
R. I. MARIO
G. BALDINI,
M.D.:
Division
of Hematologic
Research,
The
Memorial
Hospital,
Pawtucket,
R. I.;
of Medical
Science,
Brown
University,
Providence,
R. I.
Abbreviations
tidylcholine;
used
SM,
phosphatidylserine;
CER,
ceramide;
CH, cholesterol;
BLOOD,
established.
Contrary
From
the Division
Brown
University,
Submitted
January
Supported
by U.S.
tract 71 -2252.
Memorial
Division
Professor
Director,
Professor
is well
and
tidylethanolamine.
glycerides
optimal
activity
red cell storage
phospholipids.6
and
increased
requiring
the
of these
the
During
an
a
process
in platelets.
maintaining
in determining
ipate
into
a
of
by
into
From
PHYSIOLOGICAL
ponents
(2) Both
than
fatty
exceeded
reduced
al-
of platelets.
incorporated
phospholipids,
cantly
to
bound
fresh
Storage
following
palmitate
platelets.
of
linoleate
ratio of cholesterol
to phospholipids
and
percentage
distribution
of
individual
phospholipids
were
similar
in fresh
and
BALDINI
palmitate
into
incorporation
decreased
days.
Molar
6
MARIO
AND
energy.
More
was
incorporated
linoleate-1-
investigated
(4#{176}C)human
at 4#{176}C
SitINER,
MANFRED
Fatty
Platelets:
of Storage
to incorporate
palmitate-1-14C
vitro
of Free
of Human
Effect
OKUMA,
Incorporation
VoL.
38,
in text: FFA, free fatty acid; PL, phospholipid;
LPC,
lysophosphasphingomyelin;
PCH,
phosphatidylcholine;
PT, phosphatidylinositol;
PS,
PE, phosphatidylethanolamine;
PA, phosphatidic
acid; CL, cardiolipin;
CEB,
cerebroside;
MG, monoglyceride;
DG, diglyceride;
TG, triglyceride;
CHE,
cholesterylester;
TLC,
thin-layer
chromatography;
SF, solvent front.
No.
1
(JuLY),
1971
27
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28
here
defined
Lipid
as
in
peroxidation
man
vivo
survival
and
rapid
loss
of platelet
have
storage
urated
studied
and
fatty
upon
is markedly
platelets
are capable
acids.9’1
Renewal
by de novo
synthesis
fatty
we
STEINER,
OKUMA,
and
cholesterol
determined
acids
into
their
their
of Platelet
such
its outstanding
and
lost
on
conditions.8
importance
phospholipid
ability
lipids.
to
MATERIALS
Preparation
is rapidly
under
BALDINI
storage.7
Fresh
hu-
of incorporating
and metabolizing
long-chain
free
of phospholipids
by reacylation
of lysocompounds11
has been
reported.#{176}”13
In view
of the peculiarly
viability
the
reinfusion,
enhanced
AND
AND
Concentrates
and
for
content
incorporate
cell
function,
of platelets
free
saturated
during
and
unsat-
MEmsoDs
Method
of Storage
Blood
was
collected
from
healthy
male
volunteers
into
plastic
bags
( Blood pack unit
JA-2C,
Fenwal
Laboratories,
Morton
Grove,
Ill. ) containing
acid-citrate-dextrose
anticoagulant
(ACD,
U.S.P.
formula
“A”).
Platelet
concentrates
(PC)
were
prepared
and
stored
at 4#{176}C,
as descibed
previously.8
Red and white
cell contamination
was always
less
than
1 per 10,000
and
1 per 80,000
platelets,
respectively.
For incubation
with
FFA,
aliquots
of PC containing
2 X 10 platelets
were centrifuged
at 2250 g for 30 mm. The
platelet
pellet,
unless
otherwise
indicated,
was resuspended
in 2 ml of modified
Caintner’s
buffer,
pH 6.8 ( MG buffer).14
When
only the lipid content
of platelets
was to be measured,
the platelets
were washed
once with MG buffer
and
were
then
resuspended
in the
same
buffer ( 10 platelets/ml).
Separation
and
washing
of platelets
was
done
at 4#{176}C.Siliconized
glassware
was
used
throughout.
Experiments
with
fresh
platelets
were
always
started
within
5 hr from
the time of blood
collection.
Lipid
Preparations
Palmitic
acid-1-14C
53.0 mCi/mM
) were
(specific
activity:
with
the
mixed
55-57.1
respective
mCi/mM)
and
linoleic
nonradioactive
fatty
( SA:
acid-1-14C
acids
to
obtain
the
specific activities
and were then bound
to 5% bovine
serum
albumin
(BSA ) in
0.1 M Tris-HC1
buffer, pH 7.4. In case this solution
was not used promptly,
it was divided
into 0.2-ml
aliquots,
sealed
in ampules
under
nitrogen
and
stored
at -20#{176}C.14 The
desired
crystalline
BSA was assayed
for contaminating
palmitate
and linoleate
by quantitative
gasliquid
chromatography.
Lipids
were
extracted15
from
BSA-bound
palmitate
and linoleate.
After methylation,#{176} the methyl
esters were purified
by TLC and eluted
from the silica gel.16
The purified
methyl
esters
of each lipid
sample
were
subjected
to gas-liquid
chromatography.17
Appropriate
blank
areas
from the silica gel were
eluted
and carried
through
the
same procedures
as the methyl
esters.
Quantitative
analyses
of the chromatograms
proved
that
the contamination
of crystalline
BSA
with
palmitate
and
linoleate
was
negligible
compared
to the
amount
of the
respective
fatty
acid
bound
to BSA for experimental
purposes.
The purity
of all nonradioactive
lipids used was established
by TLC.
Incubation
Procedures
To 2-mi aliquots
of platelet
suspension
(2 X 109 platelets
ml of BSA-bound
fatty
acid-1-14C
(usually
5 uCi;
final
were carried
out in a metabolic
shaking
water
bath usually
95%
2-hr
washed
02
and
5%
incubation.
five
COD.
After
times
with
No
clumping
incubation
3-ml
of
the
aliquots
platelets
platelets
of
ice
was
were
cold
0.5%
in MG buffer)
were added
0.2
SA: 10 mCi/mM).
Incubations
at 37#{176}Cwith an atmosphere
of
observed
during
or at the end of a
separated
BSA
by
in
MG
centrifugation
buffer
and
at
4#{176}C,
resuspended
in 2 ml of MG buffer.
The
platelets
were
recounted’9
to correct
for their
loss
during
these
procedures.
To determine
the effect
of metabolic
or SH inhibitors
on fatty
acid incorporation into platelet
lipids,
platelets
were preincubated
for 15 mm at 37#{176}C
with the respective
inhibitor-that
is, N-ethylmaleimide
(NEM),
NaF,
or KCN
all dissolved
in MG
buffer.
Then
0.2 ml albumin-bound
palmitate-1-14C
was added
to the flasks and incubation
con-
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LIPIDS
IN
29
PLATELETS
HUMAN
tinued
for one more
hour.
The reaction
was stopped
and platelets
were
washed
as described
above.
When
the release
of lipids
from the platelets
was determined,
the platelets
were initially
incubated
with BSA-bound
palmitate-1-14C,
then washed
as described
above.
After
resuspending
the platelets
in 2 ml of MG buffer,
0.2 ml of 5% BSA in 0.1 M TrisHCI buffer
pH 7.4 was added.
Platelets
were
reincubated
for 1 hr under
conditions
described
above,
Lipid
Assays
then
resuspended
in 2 ml of buffer.
Extraction
of Lipids:
Two ml of platelet
suspension
or of incubation
medium
was extracted
with 48 ml of chloroform-methanol
(2: 1, v/v)
at room
temperature
for 30 mm.
The crude
lipid extract
was separated
after addition
of 10 ml 0.154
M NaCl.#{176}The extracts
from
platelets
incubated
with
fatty
acids-1-14C
or from
the reincubation
media
were
washed
three
times
with
chloroform-methanol-0.154
M NaC1.’5
The
extracts
were
concentrated
at reduced
temperature
in vacuo
and were
then evaporated
to dryness
under
a
stream
of nitrogen.
The residues
were
dissolved
in small
volumes
of extraction
solvent,
which
were
immediately
used
for determination
of total
radioactivity,
assay
of PL
phosphorus
and of CH,
and
for chromatographic
separations.
Determination
of Phospholipid
Phosphorus
and of Cholesterol:
PL phosphorus
was assaved
according
to Dodge
and Phillips.16
Triplicate
determinations
agreed
within
±3%
of
their
mean.
Total
CH was determined
by a modification
of Searcy’s
method.20
A small
aliquot
of the lipid
extract
was evaporated
to dryness
under
a stream
of nitrogen,
the
residue
was
dissolved
in 1 .5 ml of FeSO4 - acetic
acid reagent20
and mixed
with 0.5 ml
concentrated
HSO4.
Optical
absorbancy
was measured
after
10 mm at 490 nm. Triplicate
determinations
agreed
within
±4%
of their mean.
Separation
of Lipids:
Lipids
were
separated
by TLC
on plates
coated
with
silica
gel
N-HR
essentially
as described
before.14
Phosphorus
was assayed
in the following
spots on
the TLC
plate:
( 1 ) each clearly
identifiable
major
PL class;
(2 ) an area corresponding
to LPC;
( 3) SF, which was shown to contain
PA, CL, CER,
and CEB as well as neutral
lipids;
and (4) the origin
(it always
showed
negligible
values
of phosphorus).
Between
94 and
101 % of the PL phosphorus
applied
to the TLC
plates
was recovered
after
chromatographic
separation.
of
Assay
Radioactivity
The
radioactivity
of the original
lipid
extract
and
that
of lipids
separated
was measured
as described
previously.14
Overall
recoveries
of radioactivity
after
graphic
separation
were
approximately
96%.
by TLC
chromato-
Materials
Palmitic
acid-1-14C
and linoleic
acid-1-14C
were
obtained
from Amersham
Searle,
Des
Plaines,
Ill. Nonradioactive
fatty
acids,
SM, PCH,
PT, PS, PE,
monopalmitin,
diolein,
tripalmitin,
arachidic
acid
methyl
ester,
cholesteryl
palmitate,
CEB,
and
PA were
all
obtained
from Mann
Research
Laboratories,
New York, N.Y. CER
and CL were products
of Applied
Science
Laboratories,
State College,
Pa., and LPC was purchased
from Analab,
North
Haven,
Conn.
BHT
was
obtained
from
Sigma
Chemical
Co.,
St. Louis,
Mo.
Crystalline
BSA (all from one lot) was obtained
from Mann
Research
Laboratories,
New
York, N.Y. Silica gel N-HR
was a product
of Brinkmann
Instruments,
Westbury,
N.Y.
RESULTs
Cholesterol
Fresh
and
11.1
and
human
±
Pholwlipid
platelets
0.7
Content
from
xg phosphorus
at an almost
linear
rate
in platelets
after
6 days.
five
per
of Fresh
different
10
during
storage
The reduction
and
donors
cells
(mean
Stored
contained
±
reaching
70%
in CH and PL
SEM).
Platelets
Human
90.4
±
Both
3.2 1ttg CH
decreased
of their
initial
became
significant
content
after
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
30
OKUMA,
Table
1.-Lipid
Lipid
Phosphorus
in
Fresh
and
Stored
Fresh
Human
Stored
1 day
Total
11.05
0.71
3 days
phosphorus/b9
±
0.45
7.56
± 0.40
± 0.03
0.14
1.76
±
0.08
1.57
±
0.12
1.28
4.18
±
0.11
3.54
±
0.19
2.98
0.20
1.27
±
0.09
±
0.20
2.62
±
0.11
±
0.03
0.09
± 0.04
1.05
2.33
0.10
0.11
± 0.06
± 0.04
0.04
± 0.08
± 0.17
± 0.15
±
0.1
0.8
0.5
± 0.7
0.7
0.3
1.8
17.4
41.2
12.2
26.5
0.9
1.86
±
0.19
4.31
±
0.25
1.39
2.87
0.9
±
1.7
17.3
40.3
12.9
27.0
0.8
±
f
Percentage
LPC
SM
PCH
PT + PS
PE
SF
*
Mean
±
f p <
SEM
6 days
platelets#{176}
9.16
PCH
PS
Platelets
at4#{176}C
0.15
SM
+
BALDINI
0.49
± 0.04
P1
PE
SF
g
10.48
0.18
AND
± 0.04
±
0.18
LPC
STEINER,
±
±
±
±
of five
separate
f
§
±
§
±
0.98
1.94
0.11
±
0.10
±
0.05
±
0.2
0.4
0.3
0.5
0.5
0.2
f
§
f
§
distribution0
0.2
±
0.3
± 0.4
± 0.3
± 0.5
± 0.2
1.8
17.9
40.5
11.8
26.8
1.2
±
0.3
0.7
0.4
0.8
0.9
0.3
±
±
±
±
±
±
1.8
17.4
40.9
12.3
27.0
0.9
±
±
±
±
±
determinations.
0.05.
t p < 0.02.
§p <
0.01.
p is based
on comparison
3 days
of storage
crease
in
these
change.
stored
as
Similarly
compared
decrease
PE
be
(p
<
present
the corresponding
0.01
lipids
only
the
platelets
obtained
p <
0.02
and
thus
distribution
(Table
rate,
1 day
range.
their
and
was
uniform
after
value
symmetrical
unaffected
to fresh
in the normal
so small
and
CH
for
was
at a completely
was
still
were
with
for
PL)
platelets.
(Table
their
1).
molar
of the various
1 ). The
individual
however;
of storage
in fresh
e.g.,
while
de-
did
not
PL
classes
did
in
not
PL
a significant
total
The phosphorus
values
variations
so wide
that
The
ratio
PL
reduction
at
of LPC
and
no significant
that
( CL
SF
in
time
were
+ PA)
changes
could
observed.
In Vitro
Incorporation
of Fatty
Acids
Into
Lipids
of Fresh
and
Stored
Human
Platelets
Time
Course
of Palmitate
Incorporation
Into
Lipids
of Fresh
Platelets:
The
rate of incorporation
of palmitate
into total
lipids
was linear
during
the first
60 mm but decreased
slightly
during
the second
hour
of incubation
( Fig. 1).
Of the individual
lipid
classes
PCH
showed
the steepest
increase
and had the
greatest
fairly
total
steady
only
one
the
FFA
accumulation
rate,
but
seventh
fraction
linear
radioactivity
the
first
with
were
various
of incubation
not
lipid
(Fig.
incorporated
and
The
in the
classes
lB).
uptake
acid
at
its
figure.
The
rather
FFA
into
maximum
P1
showed
+
a
was
of palmitate
into
changed
Although
fatty
2 hr of incubation
attained
incorporation
plotted
the
after
PCH.
rapid
of palmitate
and
the
TG
accumulated
into
extremely
rates
time
among
amount
incorporated
was
The
30 mm
total
of that
instantaneously.
were
of palmitate.
the
almost
PS
and
SM
distribution
rapidly
the
of
during
highest
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LIPIDS
LX
HUMAN
31
PLATELETS
9
I
I
I
30
60
lncubojon
tim#{149} mm.
Fig. 1.-Time
course
of incorporation
of palmitate-l-14C
into lipids
of fresh
human
platelets.
(A) Absolute
incorporation
expressed
in nmoles/
l0 platelets
into
total lipids
(inset)
and various
lipid classes.
(B) Relative
incorporation
expressed
as
percentages
of total radioactivity.
PCH,
squares
with solid lines; PE, closed
circles
with
solid lines;
TG, squares
with
dashed
lines;
FFA,
closed
circles
with
dashed
lines;
DC, open
circles
with dashed
lines.
Each
point
represents
the mean
of two
experiments.
activity
initially,
major
PL
to the
total
with
its
only
PE
Release
in the
of Palmitate
comparison
the
the
Although
the sum
from
reincubation
the
Effect
level
of
Platelets:
different
from
0.09
rise,
although
for
in
classes
3%
TG
mM
was
(Fig.
which
contribution
only
the
results
the
PL
obtained
release
palmitate
platelets
in FFA
that
of
extracted
also
recovered
nonreincubated
from
on
by
Its
into
these
given.
from
sources
Incorporation
total
Approxiin
TG.
platelets
platelets,
fell
Into
platelet
at concentrations
The
rate,
was
are
and
the
significantly
platelets.
measured
3).
of fatty
acids
from platelets
is shown
in Fig.
2. For
released
into
the medium
was recovered
each
was recovered
in PCH,
DG,
and
palmitate
at a decreasing
PCh,
The
nonreincubated
Concentration
of
with
time.
Of
of the other
rapidly
the
significantly,
Platelets:
of nonreincubated
to 0.45
into
change
of the radioactivities
medium
exceeded
Palinitate
decreased
Since
albumin-bound
Incorporation
lipid
not
radioactivity
and about
radioactivities
of
below
total
figure.
From
with
values
mately
90%
of
the FFA
fraction
the
sharply.
did
shown
incubated
sum
of
increased
radioactivity
are
initially
poration
fraction
PCH
incorporation
throughout
far
the
into
this
highest
Lipids
lipids
as
of the
fatty
total
lipids
concentration
of that
into
of
Fresh
well
acid
as
continued
range.
any
into
varying
of the
to
Incorother
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OKUMA,
32
Fig.
STEINER,
AND
2.-Exchange
BALDINI
of platelet
lipids
with
incubation
medium.
Platelets
which
had incorporated
radioactive
palmitate
were
washed
and
reincubated
with
BSA-containing
buffer
without
addition
of
palmitate.
Lipids
were
extracted
both
from
the
platelets
and
from
the
reincubation
medium.
Lightly
shaded
area:
per
cent
radioactivity
in
various
lipids
of
reincubated
platelets;
mean
values
of two
experiments
are
shown.
White
area:
per
cent
radioactivity
in
lipids
recovered
from
reincubation
medium;
mean
values
of
a
0
0
a
0
0
0
C
0.
two
experiments
Heavily
shaded
radioactivity
platelets
that
bated;
mean
six experiments
SM
PCH
P1
PE
DG
FFA
are
shown.
area:
per cent
in various
lipids
of
were
not
reincuvalues
±
SEM
of
are given.
TG
PS
Fig.
3.-Effect
mitate
incorporation
of
of
concentration
fresh
palin its
into
lipids
human
platelets.
Incorporation
into
total
lipids
(inset)
and into various
lipid
classes:
PCH,
squares
with
solid
lines;
PE,
solid
closed
lines;
with
closed
dashed
circles
lines;
DC,
with
point
of
circles
with
TC,
squares
lines;
with
open
dashed
lines.
represents
the
two
experiments.
0
0
6
0
0
a
0
4
0
FFA,
dashed
E
circles
6
Each
mean
0
a
0
E
C
05
Concentration
mMP
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
LIPIDS
IN
lipid
HUMAN
classes,
33
PLATELETS
leveled
off
at 0.23
mM
palmitate.
well
The
as FFA,
increased
their
palmitate
entire
concentration
range
tested.
Effect
of Metabolic
and Sulfhydryl
Inhibitors
Platelets:
with
Preincubation
marked
PL,
reduction
TG
effect.
palmitate
Cyanide,
into
lipids.
into
FFA
of
platelets.
than
that
into
PL
nificant
platelets
in the incorporation
( Fig. 4) . Incubation
DC
and
similar
of
increase
on
None
the
other
of these
TG.
above
normal
into
DG
the
pattern
though
(p
<
0.05),
Storage
FFA
(p
in PCH
distribution
of
platelets
<
(p
0.05)
<0.01)
and
and
progressively
the
in
was
radioactivity
of incorporation
into the various
palmitate
showed
a significantly
NaF
TG
as
over
Uptake
or
the
platelets
Incorporation
of Palmitate
and Linoleate
Although
incorporation
of palmitate
and
platelets
did not differ
significantly,
each
DG,
rate
the
by Fresh
produced
a
into total
lipids
and into
in the cold
( 4#{176}C
) had a
affected
NaF-preincubated
in
NEM
enhanced
inhibitors
and
on Palmitate
either
hand,
PL
at a linear
of palmitate
of platelets
Incorporation
and
other
incorporation
of
incorporation
uptake
general
less
showed
this lipid
of
of palmitate
depressed
a highly
fraction.
sig-
Into Fresh
arid Stored
Platelets:
linoleate
into
total
lipids
of fresh
fatty
acid
showed
a very
distinct
lipid
classes
higher
TG
(p
<
PE
(p
<0.01)
decreased
0.01)
the
(Tables
incorporation
than
2 and
linoleate,
exceeded
the
that
incorporation
3).
into
AlDG
latter’s
of palmitate.
of
fatty
acids
0
C
0
0
C
6)
0.
4#{176}C
Fig.
uptake
4.-Effect
by lipids
NEM
of metabolic
inhibitors
of fresh human
platelets.
NaF
and
2
sulfhydryl-reactive
109 platelets
X
KCN
were
agent
on palmitate
preincubated
for
15 mm
at 37#{176}Cwith
either
buffer
(control),
1 mM
KCN,
10 mM
NaF,
or 1 mM
NEM.
Albumin-bound
palmitate-l-14C
was then
added
and incubation
continued
for
one more
hour.
In other
experiments,
platelets
were
incubated
with
albumin-bound
palmitate-l-’4C
at 4#{176}C
for 1 hr without
preincubation.
Incorporations
into total lipids
(white
area),
total
PL (heavily
shaded
area),
DC
(stippled
area),
FFA
(dark
area),
and TC (lightly
shaded
area) are expressed
as percentages
of control.
Mean
values
of tsvo experiments
are shown.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
34
OKUMA,
Table 2.-In
Vitro Incorporation
Human
Platelets
and Distribution
STEINER,
No.
Fresh
Total
incorporation0
(nmoles/109
platelets
4.02
SM
PCH
P1 + PS
PE
DG
FFA
TC
Mean
0
4
± 0.45
3.9
±
63.2
±
1.4
3.6
3.4
9.5
11.8
±
±
±
±
±
at 4#{176}C
3 days
1 day
6
of experiments
BALDINI
of Palmitic
Acid-b-14C
Into Fresh and Stored
of Radioactivity
Among
Major Lipid Classes
Stored
Lipid
AND
3.27
± 0.46
Percentage
4.4 ± 0.4
62.1 ± 3.0
1.2 ± 0.1
4.7 ± 0.4
3.4 ± 0.4
9.0 ± 1.1
10.5 ± 0.8
0.4
1.2
0.2
0.3
0.2
1.0
1.0
6 days
4
4
2.65 ±
distribution0
4.9 ±
56.1 ±
1.4 ±
6.6 ±
3.5 ±
9.5 ±
13.6 ±
f
0.18
0.5
0.7
0.3
0.7
0.4
1.2
1
f
0.7
2.40
±
4.2
49.8
1.7
7.9
4.9
12.6
13.9
±
0.20
0.7
3.0
0.2
0.8
0.4
2.1
2.2
±
±
±
±
±
±
f
SEM.
±
I p < 0.05.
I p < 0.02.
p is based
into
on comparison
total
fractional
were :
lipids,
(1 )
gradual
but
significant
)
and
(3
and
TG.
The
was
did
and
palmitate
incorporation
a progressive
PE;
CHE
with
the
corresponding
more
than
increase
less
for
than
PCH
Table
those
of fresh
the
of palmitate
than
1%
of
of palmitate
both
platelets;
p <
3.-In
Vitro Incorporation
man Platelets
and Distribution
0.01
into
of
both
of
linoleate
total
into
for
for
fresh
PC
of Linoleic
and
SM
PCH
PT + PS
PE
3.4
72.6
1.5
5.3
DC
FFA
TG
0
Mean
±
± 0.53
±
±
±
±
2.2
4.8
±
5.6
±
±
0.6
1.3
0.3
0.3
0.3
0.7
0.9
acids
in
MG,
into
stored
Acid-1-’4C
Into
Among
p <
Fresh
Major
and
Lipid
with
the corresponding
and
and
<
0.05
Stored
3 days
6 days
4
4
4
± 0.68
Percentage
5.5 ± 0.7
73.7 ± 3.1
1.7 ± 0.2
5.6 ± 0.3
2.4 ± 0.3
2.3 ± 0.51
5.3 ± 1.1
value
3.40 ±
distribution0
4.1 ±
75.6 ±
2.0 ±
7.2 ±
2.1 ±
1.7 ±
5.2 ±
obtained
0.05
for
Hu-
at 4#{176}C
1 day
3.95
2
Classes
0.41
2.97
0.6
0.8
0.4
0.2
0.2
0.3
0.8
5.4
75.0
2.2
7.4
2.3
1.9
2.4
1
1
SE.
on comparison
CH,
(p
platelets;
I p <0.05.
p is based
into
FFA
lipids
platelets
of stored
of Radioactivity
6
4.03
fatty
a
results
were
omitted
from
Tables
in PCH
and PE were
significantly
Fresh
No. of experiments
Total
incorporation0
(nmoles/109
platelets
( 2)
PCH;
LPC,
the
storage
linoleate
incorporation
Stored
Lipid
in
platelet
incorporation
and
These
linoleate
changes
during
accumulation
their
platelets.
principal
acids
incorporation
in
not change
during
storage.
3. The
specific
activities
of
higher
the
in fresh
The
fatty
palmitate
two
decrease
incorporation
always
in
obtained
linoleate.
of these
decline
of
a significant
value
in fresh
platelets.
±
0.45
±
0.8
2.6
0.4
0.2 f
0.2
0.3 1
0.8
±
±
±
±
±
±
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
LIPIDS
IN
HUMAN
35
PLATELETS
LINOLEATE
SM
PCH
PALMITATE
P1+ PS
PE
SM
24
PCH
P1+ PS
PE
24
20
0.
4,
0
E
-16
16
0
4)
0
t1:
8
__
4
0136
0136
0136
0136
0136
Storage
Fig.
5.-In
fresh
and
experiments
PE
vitro
stored
for
of both
1
day
(p
(p
incorporation
fresh
and
decreased
<
<
).
0.05
on
This
at
the
increased
stored
the
0.02),
PCH
of linoleate-
human
platelets.
fresh
platelets
and
reduction
which
other
specific
hand
) ( Fig.
activity
became
time
it
did
activities
and
not
in
P1
change
+
PS
5).
major
PL
are the mean
±
SEM
for stored
platelets.
into
of
Storage
of platelets
for
after
The
activity
of
linoleate
Both
fatty
acids
more
off.
0136
PCH
with
pronounced
of
leveled
0136
palmitate-1-14C
The
results
shown
of four
experiments
platelets
specific
0136
mdays
specific
during
and
respect
storage.
in
PE
when
to
3 days
platelets
of
six
only
palmitate
of storage
were
in
had
stored
3 to 6 days.
for
DIscussIoN
content
The
has
been
14.7
to
CH.26
values
of lipid
measured
23.7%
by
of the
Recalculating
ranging
from
dry
phosphorus
and
a number
weight
of
CH
in normal
investigators.232#{176}
of plate1ets3’25’27
these
fractions
78 to 126 jig
on basis
of platelet
per 10#{176}
platelets.
CH content
as measured
by us agree
as does the distribution
of lipid phosphorus
well with
among
A gradual
in red cells
content
storage
and
in
with
vitro
the
reduction
in membrane
lipid
during
in vivo aging,3#{176}during
incubation
loss
in
at
lipids
37#{176}C.31Erythrocyte
from
the
red
were
cell
viability
membranes,
fresh
human
platelets
Lipids
which
make
shown
to contain
up
19%
number
results
in CH
Total
lipid
phosphorus
the results
the different
of other
authors
PL classes.28’20
has been
reported
to occur
of blood
at 4#{176}C,#{176}
and during
decreased
although
concomitantly
the
latter
was
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
36
OKUMA,
not
considered
pattern
critical
of
similar
and
lipid
to what
total
for
loss
we
PL,
and
the
of
red
found
in
the
loss
in lipid
phosphorus
period
of storage
after
only
more
rapidly
ological
such
of
participate
Fresh
classes.9’10
FFA
equilibrated
incorporated
transfer
by NaF
rate
of
into
platelets,
that
fact
could
A distinctive
thus
( total
inhibitory
result
in
an
one
effect
be
due
to
palmitate
into
these
changes
remains
to be
acids
effect
on their
can be renewed
thesis.9’11-14
explanations
effective
in
platelets
PL,
DC,
an
number
by
lipid
the
of
incorporation
acids.10’’14
and
are causally
established.
reduced
a reduction
their
incorporation
both
by
specificity
into
stored
red
were
a decrease
cells
The
for
only
in
observed
anstim-
increased
thus
fatty
acids.
has been
shown
both
palmitate
by
the
platelets
changes
incorporation
of PE.
or simply
8 hr before
it had
that platelet
by de novo
provides
various
storage
of
Whether
coincidental
incubation
3#{216}%,35 but
shown
and
during
of
significant
amount
cells.
It has been
of lysocompounds1’
changes
rather
upon
Viability
The
into
from
TG
oxidase,
of
in viability
blood
for
in white
undeter-
eluted
from
lipids
absolute
of these
different
pathways
changed
FFA
incorporation
of the
yet
glycerophosphate,
platelets.
24 hr.
to the loss
of bovine
as
of
it can
cyanide-preincubated
sites
within
and
FFA
washing
dependent
incorporation
in the
uptake
into
acylation
of
tempera-
acids
lipids
loss
with
platelet
The
interval
sites
threefold.33’34
noted
production
in
the
fatty
the
was
esterification
extent
related
Standing
to
TG
various
pool they were
of the palmi-
of cytochrome
two-
and
decreased
time
PL
their
Extracellular
Since
to their
inhibitor
that
on magnitude
transport
of
to
of
due
factors
lowering
the
bound
physiis not
into
process.
proportion
enhanced
to a major
PCH
tightly
of potential
fatty
lipids
Operation
for the
The
an
lipid
inadequate
platelets.
remove
FFA
fraction
of platelets.
palmitate
into
TG and
into
in this
not
is probably
PL
platelets
of
FFA
of
adsorption
are
CH
all
lipid,
the
in stored
capacity
large
was
in
The rate of deWhile
a 6-wk
Although
lipids
constitute
could
The
40C6
involved
lipids
albumin-bound
a physical
FFA
glycolysis
into
found
platelets.
an
acids
Cyanide,
at 4#{176}C
is lost
fatty
of
pattern
in lipids
with
fatty
long-chain
linoleate
stored
be
were
a greater
various
to
this
a relatively
incorporation
providing
expected
solution
the
that
anaerobic
platelets
and
may
platelets
acid
of
albumin
glycolysis.
ulates
cell
cells.6
at
of lipids
occurred
in platelets
of platelets
is also lost much
in
confirm
absence
from
the original
incorporation
fatty
for
the
an
reincubated
aerobic
cells
erythrocytes.7’32
incorporate
results
which
The
The
red
BALDINI
reduction
concentrations.
quite
different.
decrease
and
a parallel
in stored
the
of
stored
rapidly
with
those
in the platelet.
From
this
into other
lipid
classes.
The extreme
rapidity
concluded
than
be
membrane
Our
with
mined.
that
AND
blood
and KCN or by sulfhydryl-reactive
compounds
acid
uptake
indicate
an energy-independent
fatty
platelets
be
than
could
platelet
and
ture,
to reduce
the
ACD
was
cellular
however,
precipitous
in loss of viability.
human
platelets
can
lipid
then
the
reduction
the
there
by
storage
of
of
30%,#{176}a similar
loss
storage
at 4#{176}C.Viability
during
yet,
skeleton
necessary
)
CH
of
meaning
clear
tate
was
original
was,
of
storage
i.e.,
in proportion
to their
in lipids
during
storage
and
6 days
survival
during
platelets;
classes
crease
phosphorus,
subsequent
cells
STEINER,
several
lipids
makes
little
PL
syn-
possible
of stored
it vir-
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
LIPIDS
IN
tually
impossible
or
HUMAN
37
PLATELETS
to indict
as a possible
a general
reduction
in necessary
transferase
or fatty
acid:CoA
in acvl
by
different
rates
of
degradation
the concentration
permeability
during
of the
storage
fatty
medium
in
acids
context
of fatty
tate
from
and
linoleate
acid
from
in aged
demonstrated
fatty
the
that
cell age was
acids
into different
incorporation
of
various
could
to
the
respect
activities
of the
the
various
in red
cells.36
Based
acids
into
ably,
as a measurement
as
platelet
in
on
lipids
enzymes
enzymes
these
could
findings
postulated
considerations
be
of platelet
as an
functions
by
alterations
that
in this
characteristics
uptake
of palmi-
red cells or their
ghosts
time there
was a decline
in red
cells
platelets,
Age
in vitro
index
are
aged
for
findings
incorporation
aging
lost
aging
by
are
with
of the
these
of cell
was
of
in vivo
particularly
dependency
to account
the
utilized
or
the incorporation
increase
in the
in stored
of palmitate.
was
of ATP
It is of importance
ligases.
These
supply
Alterations
in the membrane
for the access
of the different
plasma
into PE of human
erythrocytes.3#{176} At the same
PCH.
in the
as coenzyme
A. Changes
could
have
been produced
respective
found
to influence
lipids.
A marked
into
relevant
a decrease
substrates.
be crucial
similar
to our
observations
to the in vitro
incorporation
strikingly
well
cause
cofactors,
such
ligase
activities
of fatty
and,
prob-
in vivo
as
vitro.
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J. H., Pethica,
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A. V., and Salton,
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1955.
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1971 38: 27-38
Lipid Content and In Vitro Incorporation of Free Fatty Acids Into Lipids of
Human Platelets: The Effect of Storage at 4°C
MINORU OKUMA, MANFRED STEINER and MARIO BALDINI
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