CLIN. CHEM.23/9, 1548-1550 (1977)
PhospholipidCompositionof BloodPlasma, Erythrocytes,and
“Ghosts” in Sickle Cell Disease
Henry P. Schwarz, Miriam B. Dahike, and Lorraine Dreisbach
We examined the phospholipid composition of the plasma,
of whole erythrocytes, and of “ghosts” of the erythrocytes
from healthy volunteers, patients with sickle cell disease
without crisis, and such patients in crisis, and found that
phosphatldylglycerol
In the plasma and “ghosts” was very
significantly Increased in sickle cell crisis but not in the
absence of crisis. The significance of these findings Is
discussed.
AddItIonal Keyphrase:
phatidyiglycerol
.
crisis-associated increase In phosphosphoilpid content of erythrocytes
prostaglandins
The factors precipitating
the clinical event known as
sickle cell crisis are not clear, either to the clinicians or
to the molecular biologists. A patient with sickle cell
disease
may be asymptomatic
Presumably,
erythrocytes
for long periods
of time.
painful crisis occurs because of sickling of
within body organs. The in vivo changes
responsible
for this catastrophic
event are presumed,
but not proven, to be anoxia, increased osmolarity,
and
(or) slowing of blood flow. One would have to assume
that these factors are present at the time of crisis but
absent during the asymptomatic
interval. Data on these
variables, obtained during the crisis, are not impressive
(1). More than 25 years ago Pauling et a!. (2) suggested
that the sickling phenomenon
might be due to the
presence of a chemically different type of hemoglobin,
which
on deoxygenation
would
aggregate
into rods and
thereby twist the cells out of shape. Later it was shown
that the only difference
between
hemoglobin
S and
normal hemoglobin is that, in the former, a glutamyl
residue in position six of each beta-chain is replaced by
one valine residue (3). It has been questioned how such
an apparently minute difference, involving only two of
the 574 amino acid residues, could alter the threedimensional
structure of the hemoglobin molecule to
cause
sickling.
The possible involvement of a co-factor was stressed
recently by Johnson et al. (4), who showed that prostaglandin E2 can induce sickling in sickle cell anemia
erythrocytes
under
conditions
of reduced
oxygen
ten-
Department of Clinical Pathology, Philadelphia General Hospital,
Philadelphia, I?a. 19104.
Address correspondence
to H.P.S., at The Dorchester, Apt. 2410,
226 W. Rittenhouse Square, Philadelphia,
Pa. 19103.
Received June 18, 1976; accepted June 1, 1977.
1548 CLINICALCHEMISTRY,Vol.23,No. 9, 1977
sion. Its effect was only on intact erythrocytes
and was
not observed
in a hemoglobin
lysate. These changes
were taken as presumptive
evidence for the involvement
of the membrane in the sickling event. That the deformability of normal erythrocytes
may be decreased by
prostaglandin
E2 was shown by Allen and Rasmussen
(5), who suggested that the erythrocyte
may be the
primary receptor of prostaglandin
stimuli. Unrelated
studies of the effect of various prostaglandins
on the
phospholipids
of plasma and tissues in rats showed that
administration
of prostaglandins
to rats increased the
concentration
of certain phospholipids,
particularly
phosphatidylglycerol,
in their plasma and certain tissues
of the rats (6). It thus appeared of interest to investigate
the phospholipid
composition
of whole erythrocytes,
their membranes (“ghosts”), and the plasma of healthy
volunteers, patients with sickle cell disease but not in
crisis, and patients in crisis.
Material and Methods
We studied adult patients with sickle cell disease,
proven to be homozygous by hemoglobin electrophoresis
on cellulose acetate and proven to have normal hemoglobin content per cell and normal to high-normal cell
size as determined
by the Coulter
Model S counter.
Samples from patients were taken during asymptomatic
intervals
consisted
than 45 h
The mean
and during symptomatic
crisis; the latter
of visceral, bone, or joint pain lasting for more
and required hospital visits or hospitalization.
absolute reticulocyte counts for both groups
were the same. None of the patients
had been transfused
during the preceding six months. Controls consisted of
healthy men of ages 21, 25, and 35, and two women of
ages 21 and 40, all with a normal hemoglobin concentration, normal erythrocyte
indices, and 98% or more
hemoglobin A by electrophoresis.
The blood specimens were drawn into heparinized
tubes from the resting subjects about 4 h after the last
meal,
and
the erythrocytes
were
separated
from
the
plasma by centrifugation
for 15 mm at 1200 X g at 4 #{176}C.
The erythrocytes
were hemolyzed by adding an equal
volume of water, and the stromata
(“ghosts”) were
separated
by centrifugation
at 12000 X g and washed
thoroughly with water. Aliquots of the erythrocytes
or
plasma containing at least 100 g of lipid phosphorus
were extracted with chloroform/methanol
(2/1 by vol)
TABLE I Phospholipid Composition of Plasma, Whok Red Blood Cells and Ghosts from
Control Volunteers and Persons with Sickle Cell Disease - Without Cris,,sand in Crisis’
PLASMA
Control
CELLS
7
No.cases
lipid phosphorus
1432±68
Crisis
In Crisis
5
2062±59
7
5
7
3906±4l
3942±158
1335±
l23l±36b
1187±23’
1472±25
25±
I
27±
3
29±
351±12
Phosphatidylserine
11±
I
11±
I
17± 2’
Cardiolipin
21±
I
20± I
8± 2
Phosphatidic
acid
9± 0.3
7
1827±40’
2
182±
5
71±
198±
5
22±
I
3
80± 2
12±
I
156±3
167±5
Phosphatidylglycerol
30±
I
26± 2
38±
I”’
103±
Phosphatidylinositide
61±
2
65±
66±
3
175±11
5
37”
388±11
6
7
2547±70
951±38
983±54
5
280±
3”
261±
185±
4
138±
4
191±
6”
183± 4”
78±
2
62±
I
75± 6”
74±
2”
95± 5
143± 7”
133±
4”
149±5
114±
7
154± 9’
59±
2
51±
2
83±
5
l20±l
I
110±
9
2l8±I
I”
299±15’
62±
415±17
364±18
448±22’
272±
9
314±
8
289±
4’
679±14
662±18
649±19
411±11
Alkyl ethers
32±
2
34±
I
37±
I
149±
5
156±
3
147±
6
90±
Unknowns
39± 3
27±
2”
39± I’
73± 5
87±
7
85±
8
55± 4
c
d
e
p<O.O2. control vs sickle cell
p<O.OI, control vs sickle cell
p<O.OI, sickle cell no crisis vs sickle cell in crisis
I
p<O.O5. sickle cell no crisis vs sickle cell in crisis
g
p<O.O2. sickle cell no crisis vs sickle cell in crisis
6”’
110±
4
Values are expressed as micromoles
p<O.OS, control vs sickle cell
999±24
225±
83±
a
b
7
2653±43
340± 3’
298±
I”
I”
95±
202±
1597±58’
In Crisis
5
2540±63
Sphingomyelin
Plasmalogens
64±
2
7
4030±66
Cell Patients
Without
Crisis
In Crisis
l828±59b
Phosphatidylethanolaminc
Lecithin
Sickle
Control
Without
Without
Crisis
Total
Sickle Cell Patients
Control
Sickle Cell Patients
GHOSTS’
340±l5”
7
5”’
2
360±13’
58± 6
63±
2”
42±
46±
2
3”
per liter, mean ± SEM
for 24 h at room temperature
and in darkness. The
protein precipitates
from cells or plasma were filtered
out on sintered-glass
and the filtrates containing the
extracted lipids were evaporated
under reduced pressure at 35 #{176}C
in a stream of nitrogen.
The residue was dissolved in chloroform/methanol/
water (60/30/4.5 by vol) and this solution was passed
through a glass column packed with 2 g of Sephadex
G-25 (fine) for removal of nonlipid impurities (7). The
eluate was again evaporated in a stream of nitrogen and
the residue was dissolved in 25 ml of chloroform. The
chloroform solution was passed through a column containing about 6 g of silicic acid to remove neutral lipids,
and the phospholipids
were eluted with methanol.
Separation and analysis of the individual phospholipid
fractions was done in duplicate as described by Dawson
et a!. (8, 9). Aliquots of the just-purified
phospholipids
were subjected to mild alkaline hydrolysis with 0.03
molt liter NaOH in ethanol for 20 mm, with shaking, at
37 C, The hydrolysate was neutralized with ethyl formate and again evaporated
in a nitrogen stream. The
residue was distributed
between two volumes of isobutanol/chloroform
(1/2 by vol) and one volume of
water, and the two phases were separated by centrifugation. An aliquot of the water phase containing the
deacylated (alkali-labile) phospholipids was spotted on
Whatmann
3 MM paper and the glycerol phosphate
components
were first separated by descending chromatography
in phenol saturated
with water/acetic
acid/ethanol
(50/5/6 by vol) for 6 h. The solvents then
were removed and separation in the second dimension
was done by high-voltage
ionophoresis
in pyridine!
acetic acid buffer at pH 3.6, with a current of 100-125
mA at 2000 V for 1.5 h with a Savant instrument.
The
chromatograms
were dried and then sprayed with nmhydrin reagent to locate amino lipids and afterwards
with acid molybdate
reagent (8) to determine
the
phosphorus
compounds. The phospholipids
that were
resistant to the mild alkaline hydrolysis (plasmalogens,
sphingomyelins,
and alkyl ethers) contained
in the
chloroform phase were determined as described (9). The
individual chromatographic
spots were identified by RF
values found with deacylated authentic standards. In
the case of phosphatidylglycerol,
pure phosphatidylglycerol isolated from human plasma (10), 14C-labeled
Scenedesmus
albi cans (11) prepared in our own laboratory, or phosphatidyiglycerol
from Applied Science
Laboratories
(State College, Pa. 16801) were used.
Phosphatidylcholine,
phosphatidylethanolamine,
and
phosphatidylserine
was supplied by Calbiochem
(La
Jolla, Calif. 92037) and cardiolipin by Difco, Detroit,
Mich. 48232. Individual phospholipid
fractions were
measured by digesting the stained spots with perchioric
acid/water (720 g/liter) and subsequent determination
of inorganic phosphorus
(12, 13). The percentage
recovery of eluates from the chromatograms
of plasma
samples was 98%, from the cells, 96%, and from the
chromatograms
of “ghosts” 98.4%.
Results
Table 1 summarizes
the phospholipid
composition
of the plasma, whole erythrocytes,
and “ghosts” from
CLINICAL CHEMISTRY, Vol. 23, No. 9, 1977
1549
controls, patients with sickle cell disease not in crisis,
and patients in crisis. Results for choline plasmalogen,
ethanolamine
plasmalogen,
and serine plasmalogen
have been combined to simplify the table.
Blood plasma. The phospholipid values of the plasma
from sickle cell cases not in crisis resembled those of the
normal controls. The plasma phospholipids
from the
patient in crisis, however, showed a 46% increase in
phosphatidylglycerol,
while values for other phospholipids (lecithin, sphingomyelin)
were slightly lower, or
unchanged.
Whole erythrocytes.
Compared with the considerable
increase in phosphatidyiglycerol
in the blood plasma of
the patients in sickle cell crisis, relatively minor changes
of the phospholipid
values in whole erythrocytes
from
these patients were noted (Table 1), but will not be
discussed in detail here.
“Ghosts”.
Most of the phospholipids
of the whole
normal erythrocytes
are in the “ghosts.” About 59 to
75% of the lecithin, phosphatidylethanolamine,
and
phosphatidylglycerol
of the whole cells were contained
in the “ghosts.” In patients with sickle cell disease not
in crisis, the values for phosphatidylserine,
phosphatidylethanolamine,
and phosphatidic acid were somewhat
higher, but values for other phosphatides,
such as
phosphatidylglycerol,
were somewhat lower in “ghosts”
from asymptomatic patients. In the patient in sickle cell
crisis most of the phospholipid
values for the “ghosts”
resembled those found in the cases not in crisis. However, the phosphatidylglycerol
value for “ghosts” from
the patients in crisis showed a strikingly significant
increase of 42% and 63% as compared with figures for
the normal controls or the asymptomatic
patients.
position of the plant material was quite distinct from
that of human phosphatidylglycerol,
which showed a
relatively high concentration
of arachidonic acid not
seen in plant phosphatidylglycerol.
Because such fatty
acids as arachidonic acid are precursors of prostaglandins, our finding of increased phosphatidylglycerol
in
plasma and erythrocyte “ghosts” (membranes) appears
to be consistent with the postulated
role of prostaglandins in sickle cell disease.
We propose that this increase of arachidonic acid is
accompanied by increased prostaglandins in erythrocyte
membranes, which in turn produces aggregation of the
erythrocytes
seen in sickling.
Discussion
Biochem. J. 75,45 (1960).
Previous work on the phospholipid
composition
of
erythrocytes
failed to detect such important
components as phosphatidylglycerol,
phosphatidic
acid, or
cardiolipin. Reed et al. (18), using separation of lipids
on silicic acid impregnated
paper, had phosphatidylglycerol and other components partly contained in their
polyglycerol phosphatide
fraction. Blomstrand
et al.
(19), who combined separation of phospholipids
on silicic acid impregnated
paper by the method of Dawson
et al. (9) and phosphorus
determination
by neutron
activation, also did not detect these components.
We
believe that separation and identification may be better
by the technique we suggest in this paper.
Phosphatidylglycerol,
found to be so remarkably
increased in the blood plasma and “ghosts” of sickle cell
patients in crisis, was first identified by Maruo and
Benson (14) in chloroplasts of plants, and subsequently
was found in various species of bacteria (15), in liver
particulates (16), and in blood plasma and erythrocytes
(10). Haverkate and Van Deenen succeeded in isolating
enough of the pure compound from spinach leaves for
complete chemical characterization
(17), and Schwarz
and Dreibach
obtained chemically pure phosphatidylglycerol from blood plasma (10) of humans and determined its complete structure. The fatty acid com-
provements in the methods of determining individual phospholipids
in a complex mixture by successive chemical hydrolysis. Biochem. .1.
84, 497 (1962).
10. Schwarz, H. P., and Dreisbach,
L., Isolation and chemical char-
1550 CLINICALCHEMISTRY,Vol.23,No.9,1977
This work was supported
by Contract
from the Office of Naval Research.
No. N00014-74-A-0147-001
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