I01
Clinical Science (1982) 62,101-107
The relation between membrane cholesterol and phospholipid
and sodium efflux in erythrocytes from healthy subjects and
patients with chronic cholestasis
P. A. J A C K S O N A N D D. B. M O R G A N
Department of Biochemistry, Royal Irlfirmary,Huddersfield, and Department of Chemical Pathology. University of Leeds,
U.K.
(Receiued 10 February110 August 1981; accepted 25 August 1981)
Summary
Introduction
1. The cholesterol and phospholipid content of
the cell membrane and the efflux of sodium were
measured in the erythrocytes of patients with
chronic cholestasis and in healthy subjects.
2. The membranes from the patients contained
more cholesterol and phospholipid and had a
higher cholesterol/phospholipid molar ratio than
the membranes from the healthy subjects.
3. The sodium efflux rate constant was
reduced in the patients and this was entirely due
to a reduction in the frusemide-sensitive efflux
rate constant. There was no difference in either
the ouabain-sensitive or the ouabain plus
frusemide-resistantrate constants.
4. This reduction in the frusemide-sensitive
rate constant was associated with a reduction in
the erythrocyte sodium content.
5. When erythrocytes were loaded with
cholesterol in uifro the frusemide-sensitive efflux
rate constant was reduced by an amount similar
to that observed in the patients. In addition,
however, there was a reduction in the ouabainsensitive efflux rate constant and an increase in
the erythrocyte sodium content; neither of these
changes was observed in the patients in uiuo.
Some patients with liver disease have erythrocytes which appear as ‘target’ or ‘spur’ cells in
dried blood films i l l . Spur cells have an excess of
cholesterol in the cell membrane and target cells
have an excess of both cholesterol and phospholipid. This accumulation of lipid in the membrane
is the result of the raised concentration of lipids in
the plasma lipoproteinsin these patients [2,31.
When the erythrocyte membrane is loaded
with cholesterol its fluidity decreases [4, 51 and
this might be expected to lead to changes in
membrane permeability and sodium efflux from
the erythrocyte.
Owen & McIntyre [61 reported that sodium
efflux and efflux rate constant and the sodium
content were reduced in the erythrocytes of
patients with liver disease. The reduction in the
efflux rate constant was entirely due to a
reduction in its ouabain-resistant component and
this was inversely related to the cholesterol/
phospholipid ratio of the erythrocyte membranes.
In this study we have attempted to confirm the
reduction in the sodium efflux in erythrocytes
from patients with chronic cholestasis and further
to characterize any reduction by separately
measuring the frusemide-resistant and frusemide-sensitivecomponents of sodium efflux.
In order to test whether the observed changes
in sodium efflux could be due to the increase in
the membrane content of cholesterol and
phospholipid, we also studied the effect on
sodium efflux of loading erythrocytes with
cholesterol in uifro.
Key words: cell membrane permeability,
erythrocytes, frusemido jaundice, membrane
lipids, sodium.
Correspondence: Mr P. A. Jackson, Biochemistry
Department,
Royal
Infirmary,
Huddersfield
HD3 3EA, West Yorkshire, U.K.
0143-5221/82/010101-07$01.50/1
Q 1982 The Biochemical Society and the Medical Research Society
102
P . A . Jackson and D. B . Morgan
Subjects and methods
Lipid content and sodium eflux
Membrane lipid content and sodium efflux
were measured in erythrocytes from a total of 26
patients with chronic cholestasis and in erythrocytes from 17 healthy staff. The patients were 13
men and 13 women, aged 32-83 years, and the
staff were nine men and eight women, aged
21-53 years. All 26 patients were chronically ill
and had the typical biochemical changes of
cholestasis. Their plasma alkaline phosphatase
was between 14 and 142 King Armstrong
units/100 ml (reference range 0-13) and their
plasma bilirubin was between 38 and 404 pmol/l
(reference range 0- 17). Their conjugated
bilirubin was between 24 and 364 pmol/l. In 20
of the 26 patients the cholestasis was clearly due
to an extrahepatic obstruction, but in the other
six the cause of cholestasis has not yet been
established.
Venous blood (10 ml) was anticoagulated with
lithium heparin and centrifuged and the plasma
and buffy layer were removed.
The extraction of the membrane lipids and the
measurements of sodium efflux were started
within 1 h of the venepuncture. Erythrocytes
were loaded with 22Na by incubating 0-8 ml of
packed erythrocytes in 1.0 ml of incubation
buffer containing 50 pl ( 5 pCi) of 22NaC1solution
for 3 h at 37OC, with regular mixing. The loaded
cells were washed three times with 2 volumes of
incubation buffer. Each washing was performed
rapidly, by the use of a small high-speed
centrifuge (Beckman Microfuge) in which each
centrifugation took only 90 s. Portions (200 pl) of
the washed and loaded cells were dispersed into
aliquots (6 ml) of incubation buffer pre-warmed
to 37OC. For the measurement of total efflux rate
constant (K')the incubation medium contained
60 pl of 80% ethanol; for the ouabain-resistant
efilux rate constant (KOr)the medium contained
60 pl of ouabain solution
mol/l) in 80%
ethanol, and for the measurement of the ouabainand frusemide-resistant efilux rate constant (KO")
the medium contained 60 pl of ouabain solution
and 200 pl of a solution of frusemide (10 g/l).
Cells and incubation buffer were mixed and a
portion of the cell suspension was removed
immediately and centrifuged to provide a
measurement of the initial extracellular 22Na.The
remaining suspension was incubated at 37OC
with mixing for 1 h. Samples of the suspension
were taken at 20 min intervals and the extracellular fluid in each sample was obtained by
immediately centrifuging for 1 min in a Beckman
Microfuge. A sample of the cell suspension was
removed before and after the incubation, for the
determination of the total radioactivity. The
radioactivity was measured by dispersing 500 p1
of the supernatant or suspension in 10 ml of
scintillation liquid (Micellar Scintillator NE260,
Nuclear Enterprises Ltd) and counting the
scintillation events caused by gamma emission
from the 22Na in a Beckman LS 250 liquid
scintillation counter. Corrections for quenching
were made with an external standard and the
channels ratio method. The efflux rate constant
was calculated as the slope of the best fit linear
relation between time ( t ) and -log,[l - (supernatant counts at time t)/(suspension counts)].
The ouabain-sensitive efilux rate constant ( K O S )
was calculated as the difference between the total
emux rate constant (K') and the ouabainresistant efRux rate constant, and the frusemide-sensitive rate constant (KfS)was calculated
as the difference between the ouabain-resistant
efflux rate constant (KOr) and the ouabain- and
frusemide-resistant efflux rate constant. Sodium
efflux (M) was calculated as the product of the
efflux rate constant and the sodium concentration in washed cells.
When the loading of some of the cells with
22Na was under way, the remaining cells were
washed three times with 4 volumes of cold wash
buffer. The washed cells were centrifuged at
2200 g for 15 min at 4OC. For the measurement
of sodium content 200 p1 of the packed cells was
put in a sodium-free pre-weighed tube and the
weight of the cells was determined by the
difference. These cells were lysed by adding 10 ml
of lithium nitrate solution (15 mmol/l) and the
sodium concentration in the haemolysate was
measured by flame photometry. The concentration of sodium was calculated as mmol/kg
wet weight of cells.
For the measurement of the lipid content of the
membranes 500 pl of the washed cells was
placed in a phosphate-free tube and lysed by
adding 500 pl of deionized water [7]. The mixture
was allowed to stand for 15 min and then 5 . 5 ml
of propan-2-01 was added and the tube was sealed
and rotated for 1 h. Chloroform (3.5 ml) was
added together with 20 pl of a solution of
a-tocopherol (1 mg/l) in chloroform and the tube
was rotated for a further hour. The crtocopherol
was added to prevent oxidation of the lipids
during subsequent storage. The final extract was
centrifuged and the clear supernatant was transferred to a well-sealed tube and stored at -2OOC
for not more than 1 week until its cholesterol [8]
and total phospholipid 191 contents were
measured in duplicate. The cholesterol and
phospholipid contents were calculated per litre of
packed cells.
Erythrocyte sodium transport and membrane lipids
Cholesterol loading of erythrocyte membranes in
vitro
The method used to increase the cholesterol
content of the erythrocyte membrane was based
on that of Cooper et al. 1101. Erythrocytes were
separated from samples (20 ml) of heparinized
blood taken from healthy persons. Each sample
of cells was split into two portions. The first was
used as a control and the second was incubated
with a lipid dispersion with a high cholesterol/
phospholipid ratio in order to increase the
cholesterol content of the cell membranes. The
experiment was carried out 12 times. In order to
assess any non-specific effect of the lipid dispersion, erythrocytes from two healthy subjects were
incubated in lipid dispersions with a cholesterol/
phospholipid ratio of approximately 1, which
does not alter the cholesterol or phospholipid
content of the membranes.
The erythrocytes were washed three times with
imidazole-HC1-buffered sodium chloride solution
(150 mmol/l saline), pH 7.4 (see below). One
aliquot (3 ml) of the packed cells was mixed with
28 ml of a control mixture consisting of 24 ml of
incubation buffer and 4 ml of normal human
serum, previously inactivated at 56OC for 30 min.
Another aliquot (3 ml) of the washed cells was
mixed with 28 ml of a lipid dispersion and the two
erythrocyte suspensions were then incubated at
37OC for 18 h with continuous gentle mixing.
After the incubation the erythrocytes were
washed with the wash buffer before analysis.
The lipid dispersions were prepared by adding
24 ml of the incubation buffer to a pre-weighed
mixture of lecithin and cholesterol and sonicating the suspension for 1 h on an MSE 150 W
sonicator at maximum amplitude. The suspension
was kept cool during sonication by immersion in
a water bath. The lipid dispersion was mixed with
4 ml of heat-inactivated normal human serum
and centrifuged at 2000 g for 10 min at 4OC to
remove any suspended material which had not
dispersed.
Composition of buffers
The wash buffer contained MgCl, 104 mmol/l
and imidazole-HC1 buffer 5 mmol/l. The incubation buffer contained NaCl 140, KCl 5 ,
MgSO, 1, CaCl, 2 and glucose 10 mmolh, with
bovine serum albumin (100 mg/l). The buffered
isotonic saline contained NaCl 150 and
imidazole-HC1 buffer, 5 mmol/l. Each buffer was
made up in deionized water and was adjusted to
an osmolality of 290 f 5 mosmol/kg and a pH of
7.4 at 4OC.
8
103
Statistics
The distribution of the values in each group is
given as the mean and SD of the values. The
significance of any difference between the means
in the two groups was assessed by the t-test.
Differences in sodium transport between those
erythrocytes loaded with cholesterol in vitro and
their respective controls (unloaded cells) were
assessed by the paired t-test.
Results
Table 1 shows that the mean cholesterol and
phospholipid concentrations and cholesterol/
phospholipid ratio of the erythrocyte membrane
were all increased in the patients with chronic
cholestasis compared with the healthy subjects.
Fig. 1 shows that the increase in the cholesterol
was proportionally greater than the increase in
the phospholipid and that there was more
variability in the relationship between cholesterol
and phospholipid in the patients than in the
healthy subjects.
Table 2 shows that the intracellular sodium
and the total, ouabain-resistant and frusemidesensitive efflux rate constants for sodium were
each much lower in the patients than in the
healthy subjects (P< 0.001). The frusemidesensitive rate constant in the patients was on
average only a quarter of that in the healthy
subjects, and this reduction accounted for all of
the reduction in the ouabain-resistant efflux rate
constant. The ouabain-sensitive and the ouabain
plus frusemide-resistantconstants were similar in
the two groups. Because of the reduction in the
intracellular sodium, however, all the components
of flux were reduced in the patient group (Table
3), even if the corresponding efflux rate constant
was not reduced.
The cholesterol/phosphofpid ratio was inversely correlated with the sodium content ( r =
-0.44, P < 0.05) in the patient group but not
TABLE 1. Cholesterol and phospholipid content of
erythrocytes from 17 healthy subjects and 26 patients with
cholestasis
Mean values (withSD in parentheses)are shown.
Healthy subjects
Patients
Cholesterol
Phospholipid
(mmolh)
(mmolh)
Ratio
cholesterol1
phospholipid
3.95
(0.17)
4.61
(0.16)
(0.019)
5.08
(0.53)
5.19
(0.43)
0.980
(0.057)
P < 0.001
P < 0.001
P < 0.001
0.858
P. A . Jackson and D. B. Morgan
104
with any of the efflux rate constants, or effluxes.
Also in the Datient -gram- the intracellular sodium
was positively correlated with the frusemidesensitive rate constant ( r = +0.49, P < 0.05).
0
0
0
OO
0 8 0
oo
OW/
o /
otm
0
4
,'
1
1
5
6
Phospholipid (rnrnolh)
FIG. 1. Relation between the cholesterol and phospholipid content of erythrocytes (mmol/l of packed cells)
in.,
healthy subjects a n d o , patients with cholestasis.
The broken line is a cholesterol/phospholipid ratio of
0.86,which is the average ratio in healthy subjects.
TA8LE
Eflect of increases in the membrane cholesterol
content in vitro
Table 4 shows that the cholesterol content and
the cholesterol/phospholipid ratio were markedly
increased by incubating cells in the lipid dispeision, whereas the phospholipid content was
unchanged. This increase in cholesterol content
was associated, as it was in the patients, with a
reduction in the ouabain-resistant and frusemide-sensitive efflux rate constants. However, in
contrast to the changes observed in the patients,
the loading with cholesterol in vitro also reduced
the ouabain-sensitive rate constant, and there was
an increase rather than a decrease in the sodium
content. The increase in the sodium content was
inversely correlated with the change in K O s
(r = -0.80, P < 0.01). There was also a marginal increase in the ouabain plus frusemideresistant rate constant.
Fig. 2 shows the relationship between the
cholesterol/phospholipid ratio in. the membranes
and the percentage reduction in (a) the frusemide-sensitive rate Constant and (b) the ouabainsensitive rate constant. The cholesterol/phospholipid ratio was significantly and inversely cor-
2. Erythrocyte sodium content and the sodium efluux rate constants in the healthy subjects and the patients with
cholestasis
EfRux rate constants: K', total; KOr, ouabain-resistant; KOs, ouabain-sensitive; KOr*, ouabain and frusemide-resistant; K",
frusemide-sensitive. N.S., Not significant.
~~
~
~~
EfAux rate constants (h-I)
Healthy subjects
Mean
SD
No.
Patients
Mean
SO
No.
Sodium content
(mmollkg)
K'
6.47
I .08
17
0,428
0.068
17
0.105
0.323
0.065
17
0.052
0.030
17
5.79
1.13
26
0,355
0.09 I
26
0.067
0.023
26
0.287
0.081
26
0.059
0.021
17
0.013
0039
17
P < 0.05
P < 0.005
P < 0.001
N.S.
N.S.
P < 0.001
K
KO"
0'
KO"
0.015
16
K"
0.049
0.017
16
TABLE 3. Sodium efluxes in the healthy subjects and the patients with cholestasis
Sodium efflux: M', total; M"', ouabain-resistant;M"",ouabain-sensitive;Mu",
ouabain and frusemide-resistant; M", frusemidesensitive. N.S., Not significant.
Sodium emux (mmol h-' kg-')
Healthy subjects
Mean
SD
No.
Patients
Mean
SD
No.
M'
M Or
M06
MOlC
M"
2.786
0.378
17
0.666
0.195
17
2.099
0.362
17
0.339
0.101
16
0.318
06110
16
2.06
0.417
26
0.388
0. I56
26
1.672
0.321
26
0.349
0.121
17
0.086
0.064
17
P < 0.001
P < 0.001
P < 0.001
N.S.
P < 0~001
105
Erythrocyte sodium transport and membrane lipids
Effects of loading of 12 samples of erythrocytes with cholesterol in vitro on the mean cholesterol and phospholipid
content of the membranes (mmol/I),on the sodium content of the erythrocytes and on the sodium e f l u rate constants
Abbreviations are as identified in Tables 1 and 2.
TA0LE 4.
Cholesterol Phospholipid
(mmolll)
(mmol/l)
Efflux rate constant (h-')
Ratio
cholesterol1
phospholipid
Sodium
content
(mmollkg)
K'
K"'
K 0'
K"
KO*
Controls
(unloaded
cells)
3.80
4.47
0.85
4.6
0.360
0.105
0.256
0,047
0.057
Loaded
cells
6.67
4.49
1.48
6.5
0.267
0.073
0.193
0.050
0.024
P < 0.01
A
t
1
1
1
< 0.001
-4
la1
m
0
P < O O J I P < 0401 P < 0401 P < 0.005 P
1
0 8
l
*
l
*
l
l
l
13
1
18
Ratio cholesterollphospholipid
0
I
0 8
l
l
l
l
l
l
l
l
l
13
l
18
Ratio cholesteroVphospholipid
FIG. 2. Relation between the cholesterol/phosphotipidratio of cholesterol-loadedcells and (a) the
frusemide-sensitiveefflux rate constant (K ") and (b) the ouabain-sensitive rate constant ( K O s ) .
The values of the rate constants in the loaded cells are expressed as percentages of the values in the
same cells not loaded with cholesterol. 4 Mean value in uiuo in the healthy subjects; 0,mean
value in the patients with cholestasis.
< 0.05)but not to
disease. Our more detailed studies show that this
reduction in the ouabain-resistant efflux rate
constant was entirely due to a reduction in its
frusemide-sensitive component. There was no
Discussion
significant change in either the ouabain-sensitive
rate constant (the sodium pump) or the ouabain
The fluidity of the cell membrane varies with its
plus frusemide-resistantefflux rate constant.
cholesterol and phospholipid content [ 111 and
The rate constants and cell sodium we
membrane fluidity is regarded as an important
determinant of the activity of the (Na+ + K+)- measured in healthy subjects agree well with
ATPase 12-141. The non-energy-requiring those reported by Owen & McIntyre [61, except
that our values for the ouabain-sensitive rate
transport of sodium through the membrane might
constant are slightly higher than theirs. However,
also be expected to be affected by the tighter
our values are in good agreement with the other
packing within a lipid bilayer loaded with
values reported in the literature [151.
cholesterol.
When erythrocytes from healthy subjects were
The erythrocytes of our patients with
cholestasis had a reduced sodium content and a
loaded with cholesterol in vitro there were
reductions of the ouabain-resistant and frusereduced sodium efflux and efflux rate constant.
mide-sensitive efflux rate constants which were
The reduction in the efflux rate constant was due
similar to those observed in our patients with
to a reduction in the ouabain-resistant efflux rate
cholestasis. The reduction in K f s was present at
constant. These findings confirm those reported
by Owen & McIntyre [61 in patients with liver
the lowest cholesterol/phospholipidratio and did
related with KOs ( r = -0.62, P
K".
106
P. A . Jackson and D. B. Morgan
not fall further with increased cholesterol loading.
Wiley & Cooper [ 161 also demonstrated a nearly
complete inhibition of frusemide-sensitive sodium
flux in human erythrocytes which had been
enriched with cholesterol in vitro. These findings
suggest that the changes in the frusemide-sensitive sodium transport system in our patients with
cholestasis are due to the increase in either the
cholesterol content or in the cholesteroVphospholipid ratio of the erythrocyte membrane.
Wiley & Cooper [171 showed that frusemide
inhibited the sodium influx as well as efflux in
erythrocytes and suggested that there was a net
influx of sodium through the frusemide-sensitive
route. Our patients with cholestasis had a
reduced cell sodium, which might therefore be
due to the reduced frusemide-sensitive rate
constant, particularly as the two changes were
statistically related. However, if the relationship
between sodium efflux and sodium content was
sigmoid, the reduced influx itself [6] would lead to
reduction in the sodium content and in the K".
The cells loaded with cholesterol in vitro had a
raised sodium content due to a fall in K O s . Despite
this higher sodium content they had a reduced
K". This indicates that an increase in membrane
cholesterol/phospholipid ratio does decrease the
frusemide-sensitive efflux as well as the influx and
the net effect is a reduction in the sodium content.
Owen et a f . 1181 have reported that the
mechanism of the raised membrane cholesterol/
phospholipid ratio in liver disease is probably
different in obstructive jaundice and parenchymal
liver disease without cholestasis, and there may
be differences in the frusemide-sensitive sodium
transport between the two conditions. However,
all of our patients had cholestasis.
The clinical implications of the observed
changes in sodium transport in cholestasis are
difficult to assess, as the physiological role of the
frusemide-sensitive transport system in mammalian cells is not known. The general implication is, however, that changes in membrane
fluidity due to lipid accumulation may also cause
changes in the functioning of receptors or carriers
within the membrane and that this might contribute to some of the metabolic disturbances of
liver disease.
When erythrocytes from healthy subjects were
loaded with cholesterol in vitro there was a
reduction in the ouabain-sensitive efflux rate
constant which was not seen in our patients with
cholestasis and which was related to the increase
in the cholesterol content of the membranes.
Cooper et a f . [lo] concluded that loading of
erythrocytes with cholesterol in vitro did not
change the ouabain-sensitive efflux. On the other
hand, Giraud et al. 1191 suggested that in some
circumstances cholesterol reduced active sodium
efflux, and in others it increased the pump's
affinity for intracellular sodium and thereby
increased active sodium efflux. The reduction in
the ouabain-sensitive rate constant in the
cholesterol-loaded cells but not in the patient's
cells (in vivo) may be explained by the extent of
the increase in the cholesterol/phospholipid ratio
in the two states. On average the erythrocytes
from the patients had a 25% increase in membrane cholesterol, but because of a rise in
phospholipid content the cholesterol/phospholipid ratio increased only from 0.86 to 0-98.
Erythrocytes loaded in vitro showed little change
in phospholipid content so that, although the
increase in the cholesterol content was about the
same as in the patients, the cholesterol/
phospholipid ratio was higher (between 1 - 1 and
1.8). Sodium pump activity may therefore be
more dependent on the cholesterol/phospholipid
ratio than on the cholesterol content only. We
cannot, however, exclude the possibilities that
excess cholesterol may affect pump activity in
more than one way, as suggested by Giraud et a f .
191, or that excess cholesterol may be located
differently within the membrane in uivo than in
vitro. Thus addition of cholesterol to the
phospholipid environment of isolated (Na+ +
K+)-ATPase causes inhibition of its activity,
which suggests that cholesterol is excluded from
the pump sites of intact membranes [ 13,201.
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