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Effect of Uremia on the Membrane Transport Characteristics
of Red Blood Cells
By Leah J. Langsdorf and Andrew L. Zydney
Even though there is extensive evidence that uremia affects
the fragility and deformabilityof red blood cells (RBCs), essentially all data on the RBC membrane permeability have
been obtained with nonuremic blood. Permeability data
were obtainedfor creatinine and uric acid, two metabolites
of interest in hemodialysis, using a stirred ultrafiltrationdevice with direct cell- and protein-freesampling. Experiments
examined the effects of temperature and suspending phase
on solute transport for both normal and uremic blood cells.
Creatinine and uric acid transport from normal RBCs at
37°C were characterized by saturation half-times of 40 f
10 minutes and 54 f 1 2 minutes, respectively. The cor-
responding half-times for uremic cells were significantly
longer, 94 k 26 minutes and 1 8 0 rtr 38 minutes. Data indicated that the slower rate of creatininetransport in uremic
blood was caused by an alteration in the RBC membrane,
while the reduction in uric acid transport was associated
with alterations in the uremic plasma. The temperature dependence of the RBC permeability was also much less pronounced for uremic cells for both solutes. These results
provide important insights into the effects of uremia on the
RBC membrane permeability, and have important implications for dialysis.
0 1993 by The American Society of Hematology.
T
mic patients, which could have contributed to the observed
reduction in RBC deformability. There was also a significant
alteration in the lipid composition of the cell membrane during dialysis, including an increase in cholesterol and a decrease
in sphingomyelin, two structural components of the membrane. These results indicated that a significant exchange of
lipids occurred between the plasma and RBCs during dialysis,
even though there was no observable effect on the RBC deformability.
Perez-Fontan et a14 examined the deformability of RBCs
from uremic patients on both hemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD). Cells from
both HD and CAPD patients showed reduced deformability,
both in autologous plasma and in phosphate-buffered saline
(PBS), with the reduction in deformability being greater for
HD patients. Transfer of the uremic cells to a nonuremic
buffer (Hanks’ medium) caused a marked reduction in mean
corpuscular volume and a corresponding increase in the mean
corpuscular hemoglobin concentration, which may have been
associated with improved erythrocyte membrane function
on removal from uremic plasma.
However, there have been very few investigations of the
effect of uremia on the RBC membrane permeability. Skalsky
et al’ obtained limited data for the transport of I4C-labeled
creatinine across the RBC membrane for both uremic and
nonuremic cells. The equilibrium partition coefficient was
unaffected by uremia. However, the RBC membrane permeability of uremic cells was significantly less than that of
nonuremic cells, which the investigators attributed to an alteration in the RBC membrane associated with uremia.
In contrast to Skalsky’s results, Fervenza et aI6 found that
the transport of I4C-labeled lysine was significantly greater
for cells from uremic patients than for normal cells. Fervenza
et a1 provided no discussion of the origin of the markedly
different behavior seen in their study and that of Skalsky et
al.’ However, lysine is transported by the y+ carrier system6
while creatinine transport is by passive diff~sion.~
In addition,
Skalsky et a1 performed their experiments using RBCs suspended in autologous plasma, while Fervenza et a1 performed
their experiments using washed RBCs resuspended in isotonic
saline (primarily NaCl). The possible alteration in the properties of uremic RBCs on transfer to nonuremic medium was
described by Perez-Fontan et a14 as discussed above.
HERE IS extensive experimental evidence that uremia
affects red blood cell (RBC) mechanical properties such
as deformability and fragility. Forman et all found that RBC
deformability, evaluated from measurements of the filtration
half-time through polycarbonate filters, was significantly reduced for dialysis patients who had been previously splenectomized, but was essentially normal for nonsplenectomized
dialysis patients. RBC deformability was improved after a
dialysis session, suggesting that the uremic toxins responsible
for the reduced deformability could be removed by hemodialysis. Rosenmund et a12 evaluated the filtration half-time
with a more sensitive small pore filter and observed a reduced
cell deformability in almost all uremic patients, with the osmotic fragility and the sulfhemoglobin provoked by oxidative
stress also being elevated in these patients. Rosenmund et a1
hypothesized that these rigid cells were more readily removed
by the spleen, with this increased splenic sequestration contributing to the anemia associated with end-stage renal disease.
The very large reduction in RBC deformability in the splenectomized uremic patients studied by Forman et all may
have been associated with the increased number of rigid cells
remaining in the circulation in the absence of this splenic
sequestration.
Peuchant et a13 also found a marked reduction in the deformability of erythrocytes obtained from hemodialysis patients. Data indicated that there were no differences in RBC
deformability before and after dialysis, in contrast to the results reported by Forman et al’ for cells from splenectomized
patients. Peuchant et a1 also found that the cholesterol content
in the erythrocyte membrane was elevated in cells from ureFrom the Department of Chemical Engineering, University qfDelaware, Newark.
Submitted June 2, 1992;accepted October 1, 1992.
Supported in part by grantsfvom Baxter Healthcare Corp, Delaware
Research Partnership Program, and by a National Science Foundation
Graduate Fellowship for L.J.L.
Address reprint requests to Andrew L. Zydney, PhD, Department
of Chemical Engineering, University ofDelaware, Newark, DE 19716.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 I993 by The American Society of Hematology.
VVV6-4971/93/8103-VV09$3.VV/O
820
Blood, Vol 81, No 3 (February 1). 1993: pp 820-827
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82 1
EFFECT OF UREMIA ON RBC MEMBRANE TRANSPORT
Thus, despite the fact that the permeability of uremic cells
is a significant factor governing the overall rate of solute removal during hemodialysis, there is still considerable discrepancy regarding the effects of uremia on solute transport
across the RBC membrane, and there is currently no fundamental understanding of the biochemical and/or biophysical basis for these effects. Furthermore, all of the described
studies were performed before the clinical introduction of
the recombinantly produced human hormone erythropoietin
(Epo), which is now used extensively in almost all dialysis
units. Most kidney failure patients suffer from anemia due
in large part t o the reduction in Epo production associated
with renal disease. Clinical therapy with Epo has dramatically
reduced the anemia associated with end-stage renal disease
and has in many cases eliminated the need for blood transfusions in these dialysis patients. There are currently no data
available on the possible effect of Epo administration on the
RBC membrane permeability.
The objectives ofthis work were: (1) t o obtain quantitative
data for the transport of creatinine and uric acid for RBCs
from hemodialysis patients (most of whom were treated with
Epo); ( 2 ) to compare the results for uremic cells with data
for normal blood cells; and (3) to examine the underlying
biochemical and biophysical basis for the effects of uremia
on the RBC membrane permeability.
MATERIALS AND METHODS
Experimental procedures. Small samples (approximately 5 mL)
of uremic blood, collected in ACD anticoagulant, were obtained from
31 hemodialysis patients (15 male and 16 female) at Bryn Mawr
Hospital (Bryn Mawr, PA) and from 32 hemodialysis patients (16
male and 16 female) at Lankenau Hospital (Wynnewood, PA). Samples were collected monthly immediatelybefore the dialysis sessions,
with the samples from a given location collected over a 2-day period
and then pooled by blood type before use. Table 1 contains statistical
information on the two patient populations and the dialysis specifications. All patients from Bryn Mawr were treated with Epo, while
only two thirds of the patients from Lankenau were on Epo therapy.
Normal blood was collected in CPD-A 1 anticoagulant by the Blood
Bank of Delaware (Newark, DE). After the uremic blood was pooled,
all storage and preparation procedures for the uremic and normal
blood were identical. All blood was stored at 4"C,with the experiments
performed within 7 days of the initial blood collection.
The RBC permeability was measured for: (1) normal and uremic
cells suspended in autologous plasma; (2) normal cells washed and
then resuspended in uremic plasma; and (3) normal and uremic cells
suspended in PBS. PBS was prepared by dissolving 8.04 g
Na2HPO4*7H20, 4.08 g KH2P04,0.54 g adenine, 29.68 g dextrose,
and 0.67 g NaOH in sufficient deionized distilled water to make one
liter of solution. The pH was adjusted to 7.40 ? 0.05 with either
NaOH or HCI, and the osmolarity was adjusted to 297 k 3 with
either dextrose or water. For experiments in which PBS was the suspending medium, the cells were first washed two times with PBS by
centrifuging at 1,200 to 1,400g for 9 minutes. The cells were then
incubated overnight in PBS at 4°C at a hematocrit of approximately
0.4. A similar procedure was used for the experiments employing
normal cells in uremic plasma, with the uremic plasma collected by
centrifugation of the pooled uremic blood samples. In each case, the
cells were allowed to equilibrate at the experimental temperature for
I hour before the start of the experimental run.
Creatinine and uric acid RBC permeabilities were evaluated simultaneously using a stirred ultrafiltration device (Amicon Corp,
Beverly, MA). Approximately 50 to 80 mL of plasma or PBS, containing both creatinine and uric acid at concentrations between 6
and 18 mg/dL, were initially placed in the stirred device. The plasma
experiments actually used a mixture of plasma with PBS in a 4.1
ratio, with the PBS prepared with 40 to 90 mg/dL of both creatinine
and uric acid to achieve the desired final concentrations. Twentyfive to 50 mL of blood (at a hematocrit of about 0.8) were then added
to the device and the device stirrer was set at 200 to 250 rpm. Celland protein-free samples were collected periodically through a semipermeable 30,000 molecular weight cut-off polyethersulfone membrane (FiltronCorp, Northborough,MA) by air pressurizingthe stirred
cell. Additional details on the apparatus are provided el~ewhere.'.~
All experiments were performed with solute flux into the RBCs; a
previous investigation using the same experimental apparatus and
solutes showed that the influx and efflux experiments gave essentially
Table 1. Patient Statistics and Dialysis Specifications
Bryn Mawr Hospital
Patient age (yrs)
Weight (kg)
Time on dialysis (mos)
Hematocrit
Sessions per week
Length of session (h)
Dialyzers used
Blood flow rate (mL/min)
Dialysis flow rate (mL/min)
Dialysate
Epo dosage (U/treatment)
Pre-dialysis blood urea nitrogen (mg/dL)
Pre-dialysis creatinine (mg/dL)
Pre-dialysis uric acid (mg/dL)
Pre-dialysis potassium (mg/dL)
Pre-dialysis phosphorus (mg/dL)
Lankenau Hospital
Average
Range
Average
Range
60
63.5
24
0.30
31-81
35-126
3-52
0.27-0.44
70
70
18
0.28
20-82
37-95
1-168
0.20-0.34
3
3.5
Baxter cellulosic
300
500
Bicarbonate: 20 patients
Acetate: 1 1 patients
2,000-16,000 (all 3 1 patients)
61
8.4
5.6
4.3
4.9
3
3
Baxter cellulosic and Fresenius polysulfone
400
500
Bicarbonate:all 32 patients
4,000-10,000(21 of 32 patients)
50
10
2-5
5.O-5.5
4.5-7.0
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822
LANGSDORF AND ZYDNEY
identical results for the RBC membrane permeability.* Creatinine
and uric acid concentrations were measured spectrophotometrically
using commercial diagnostics (Sigma Chemical, St Louis, MO) with
an accuracy of 0.1 mg/dL for both solutes. Experiments were performed at room temperature (23"C), 30°C, and 37°C. For experiments at 30 and 37"C, the stirred cell was heated by wrapping the
device with heating tape, with the temperature controlled to within
1"C using a Digi-Sense temperature controller (Cole-Parmer,Chicago,
1L).
RBC morphology was examined by placing a small sample of diluted RBCs (in the appropriate suspending medium) on a glass slide,
which was then covered with a glass cover slip and observed microscopically with a Leitz microscope (Leitz, Malvern, PA) at 312.5X
power. RBC lysis was checked by visual inspection of the supernatant
obtained by centrifuging a small blood sample collected directly from
the stirred device at the end of the experiment. RBC concentrations
were determined using the microhematocrit method by centrifugation
of small blood samples at 2,OOOg for 30 minutes.
Data analysis. The RBC membrane permeability was calculated
by assuming that solute transport across the RBC membrane was
proportional to the effective concentration driving force:
where J is the solute flux across the membrane in units of g/cm2/s,
k is the membrane permeability, and Cf and C, are the fluid and cell
phase solute concentrations. At long times, the solute concentrations
in each phase attain their equilibrium values, but because of the
differences in the chemical nature of the cell and fluid phases, these
equilibrium concentrations are unequal. The equilibrium ratio ofthe
solute concentration in the cells to that in the fluid is defined as the
equilibrium partition coefficient, I&,which was determined experimentally as I& = Cc(final)/Cf(final),where C,(final) and Cf(final)
are the equilibrium (final) solute concentrations in the cell and fluid
phases, respectively. Fluid phase resistance to transport was assumed
to be negligible because of the bulk stirring. The internal resistance
within the cell was also assumed to be negligiblebecause of the cell's
small size as discussed by Colton et a1.l' The overall solute mass
balance on the fluid phase is thus given as:
initial blood sample before the experiment. The initial fluid phase
concentration was then determined from the known concentrations
and volumes of the plasma (or PBS) initially in the stirred cell and
the fluid phase in which the blood cells were suspended. The best fit
value of the permeability was determined numerically by minimizing
the sum of the squared residuals between the experimental and calculated values of the fluid phase concentration as a function of time
for each run using the method of steepest descent.
RESULTS
Creatinine transport. Typical data for the variation of
the creatinine concentration in the fluid phase over the course
of an expenmental run are shown in Fig 1 for both normal
and uremic cells at 37°C. The error bars represent the standard deviation in the concentration measurements, all of
which were performed in triplicate. The solid lines were evaluated from Equation (3) with the best fit value of the permeability determined by comparison of the model and data.
The good agreement between the model fits and the experimental data over the entire time course of the experiment
indicates that Equation (1) provides an accurate description
of creatinine transport under these experimental conditions.
Microscopic observations showed that the cells suspended in
plasma were discocytic in shape, while a small percentage
(-5%) of the RBCs suspended in PBS were echinocytic. RBC
lysis was not significant. The creatinine concentration in the
fluid phase decreased with time as creatinine was transported
across the RBC membrane and into the RBCs. The creatinine
concentration decayed significantly more rapidly for the normal cells than for the uremic cells, indicating that the RBC
membrane permeability to creatinine was significantly reduced in the uremic cells. The equilibrium partition coefficient for normal cells suspended in normal plasma at 37"C,
evaluated from data taken at very long times in separate experiments, was & = 0.72 -t 0.05, in good agreement with
where Vf and V, are the fluid and cell volumes, respectively, and a
is the specific surface area, which is equal to the cell surface area
divided by the cell volume. The specific surface area has been determined from microscopic observations by Buzdygon' as a = 17,000
cm-' for RBCs in the 4:1 plasma:PBS mixture used in this study,
and as a = 14,000 cm-' for cells in PBS. Assuming that the cell
properties (k, a, I&)are independent of time and concentration,
Equation (2) can be integrated to yield:
with CJt) related to Cf(t) using an overall mass balance:
v,c, + VfCf = VcCe0+ VfCfO
0
(4)
where the subscript "0" denotes the values at the start of the experiment. The initial solute concentration within the cells, Cd, was evalwhere C,, the solute concentration ofthe suspending
uated as &C,,
medium in which the cells were equilibrated, was determined from
a small sample of the supernatant obtained by centrifugation of the
1800
3600
5400
7200
9Mx)
10800
Time, t (sec)
Fig 1 . Normalized creatinine concentration in the fluid phase as
a function of time for both normal and uremic cells in autologous
plasma at 37°C. Solid lines are model correlations with k = 22.3
X
and 8.3 X
cm/s for the normal and uremic cells,
respectively.
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EFFECT OF UREMIA ON RBC MEMBRANE TRANSPORT
data obtained in previous st~dies.~,'
&I for uremic cells was
assumed to be equal to that for normal cells, as suggested by
limited data for the creatinine equilibrium partition coefficient obtained with uremic cells.
The best fit values for the creatinine permeability of both
normal and uremic cells at 37°C are summarized in the first
part of Table 2. The results are reported as the mean f standard deviation for data obtained in multiple runs (with the
number of runs given in parentheses). For normal cells, the
permeability was measured for cells from individual donors,
thus the standard deviation in the permeability reflects both
experimental errors and donor to donor variations. For the
uremic cells, the permeability was measured for cells from a
large number of pooled donors; thus, the standard deviations
are primarily a measure of the experimental errors involved
in evaluating the permeability from the solute concentration
data. There was no statistical difference in the measured permeabilities for the different pooled samples (ie, different blood
type groups) for the uremic cells, indicating that the observed
RBC permeabilities are representative averages for the dialysis
populations examined in these studies. The best fit values of
the permeability were independent of the initial fluid phase
concentration over the range of concentrations examined experimentally. The creatinine permeability for normal cells in
normal plasma at 37°C corresponds to a saturation half-time
(t,,2)of 40 f I O minutes, where t1,2is defined as In (2)/ka.
The creatinine permeability for uremic cells in uremic plasma
at 37°C was significantly less than that obtained for normal
cells in normal plasma (P< .OO 1 as determined by a Student's
t-test). The saturation half-time for the uremic cells was equal
to 94 f 26 minutes, more than twice the value obtained with
the normal cells.
To examine the origin of these differences in permeability
between normal and uremic blood, creatinine transport data
were obtained with uremic and normal cells suspended in
PBS and with normal cells suspended in uremic plasma. The
equilibrium partition coefficient for cells suspended in PBS
was not significantly different from that for cells in plasma
(0.74 f 0.09 v 0.72 f 0.05). The creatinine permeability of
normal cells in PBS was approximately three times greater
than that for uremic cells in PBS at 37°C (P< .O I), analogous
to the behavior seen for the normal and uremic cells in autologous plasma. In contrast, the creatinine permeabilities
for normal cells in normal and uremic plasma were essentially
identical. Thus, the effect of uremia on the RBC membrane
permeability to creatinine appears to be caused by a direct
alteration in the transport characteristics of the uremic cells,
with the uremic plasma itself having no significant effect on
the observed permeability.
A single experiment was also performed to evaluate the
creatinine permeability for uremic cells suspended in normal
plasma, with the uremic cells incubated in the normal plasma
at 37°C for 4 hours before the start of the experiment. The
creatinine permeability of these uremic cells was comparable
with that for normal cells in normal plasma (19.6 f 3.6 X
v 16.7 f 3.8 X
cm/s). This 4-hour incubation of
the uremic cells in normal plasma increased the RBC membrane permeability by more than a factor of two, from 7.2
f 2.0 X
to 19.6 f 3.6 X
cm/s, completely reversing
823
the effect of uremia on the permeability. Thus, the alteration(s) in the RBC membrane responsible for the reduced
creatinine transport of the uremic cells appears to be reversible
on incubation of these RBCs in normal (nonuremic) plasma
but not in (nonuremic) PBS, at least over the time periods
used in this study.
Uric acid transport. Typical data for the variation of the
uric acid concentration in the fluid phase over the course of
an experimental run are shown in Fig 2 for both normal and
uremic cells at 37°C. The equilibrium partition coefficient
for normal cells in normal plasma was evaluated as 0.52 k
0.07, with the value for uremic cells again assumed to be
equal to that for the normal cells. The solid lines were again
evaluated from Equation (3). The results are in very good
agreement with the experimental data over the entire time
course of the experiment, indicating that Equation ( I ) provides an accurate description of uric acid transport, even
though uric acid is transported by a carrier-mediated mechanism." This reflects the relatively low uric acid concentrations used in these experiments, all of which were well below
the Michaelis-Menten constant (KM = 50 mg/dL) for uric
acid transport.' I
The best fit values for the RBC permeability to uric acid
are summarized in Table 3. The uric acid permeability for
uremic cells in uremic plasma was significantly smaller than
that for normal cells in normal plasma at 37°C (P< .001),
which is similar to the behavior seen with creatinine. The
saturation half-times for normal cells was 54 +- 12 minutes,
while that for uremic cells was I80 f 38 minutes, more than
three times the value obtained with the normal cells.
Uric acid transport data were also obtained with both uremic and normal cells suspended in PBS and with normal
cells suspended in uremic plasma. The &I values in PBS
and plasma were again the same. In contrast to the results
with creatinine, the uric acid permeability for uremic cells
in PBS was essentially identical to that for normal cells in
PBS. However, the permeability for normal cells in uremic
plasma was significantly less than that for normal cells in
normal plasma at 37°C (P< .01). In addition, the uric acid
permeability for uremic cells in normal plasma (incubated
at 37°C for 4 hours before the start of the experiment) was
comparable with that for normal cells in normal plasma ( I 3.8
v 12.2 f 2.7 X
f 3.2 X
cm/s). These results clearly
indicate that the effect of uremia on the uric acid permeability
is associated with an alteration of the plasma and is largely
unaffected by the properties of the cell, exactly opposite the
behavior seen with creatinine.
Temperature effects. Experimental data for K, as a
function of temperature are given in Table 2 (for creatinine)
and Table 3 (for uric acid) with the results reported as the
mean f standard deviation for data obtained in multiple
runs (with the number of runs given in parentheses). I(eq for
creatinine was independent of both temperature and suspending phase. In contrast, Keq for uric acid increased slightly
with decreasing temperature, with no statistically significant
differences between the values obtained in plasma and PBS
at any temperature. This temperature effect is discussed in
more detail later. The equilibrium partition coefficient for
creatinine was taken as kg= 0.73 at all three temperatures
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824
LANGSDORF AND ZYDNEY
while that for uric acid was taken as 0.52, 0.62, and 0.72 at
37, 30, and 23"C, respectively.
Experimental data for the RBC permeability of normal
and uremic cells as a function of temperature are also given
in Table 2 (for creatinine) and Table 3 (for uric acid). The
data for uremic cells at 23°C were obtained with blood samples from Bryn Mawr while the data for uremic cells at 30
and 37°C were obtained with samples from Lankenau. However, a set of experiments performed at 23°C with samples
from Lankenau showed no statistical differences in either the
creatinine or uric acid permeabilities for blood obtained from
the two centers, indicating that the slight differences in the
patient populations, dialysis procedures, and/or blood acquisition methods shown in Table 1 had no significant effect
on the measured permeability.
The uric acid and creatinine permeabilities for normal cells
in normal plasma decreased with decreasing temperature,
with the temperature dependence being somewhat greater
for uric acid transport. The creatinine permeability for uremic
cells in uremic plasma was significantlyless than that obtained
for normal cells in normal plasma at all temperatures studied
( P < .001 at 37"C, P < .05 at 30"C, and P < .05 at 23°C as
determined by a Student's t-test). The magnitude of the difference in the permeability between the normal and uremic
cells decreased with decreasing temperature, with the ratio
of the two permeabilities decreasing from 2.3 at 37°C to 1.3
at 23°C. In contrast, the uric acid permeability for the uremic
cells at 23°C was actually somewhat greater than that for the
normal cells (P< .15), which is exactly opposite the behavior
seen at both 37" and 30°C.
The influence of temperature on the RBC permeability
for normal and uremic cells, each in autologous plasma, is
1.00
*
"""
e
O'*O
0.75
t
T
\Iuremic cells
1
h normal cells
1
t1
0
1800
3600
5400
7200
9000
10800
Time, L (sec)
Fig 2. Normalized uric acid concentration in the fluid phase as
a function of time for both normal and uremic cells in autologous
plasma at 37°C. Solid lines are model correlations with k = 12.5
X
and 3.8 X IO-$ cm/s for the normal and uremic cells, respectively.
shown in more detail in Fig 3 for creatinine (top panel) and
uric acid (bottom panel) with the logarithm of the permeability plotted against the inverse of the temperature (scaled
by the ideal gas constant, R = 1.987 cal/mol OK). The permeability increased with increasing temperature for both
uremic and normal cells. However, the rate of increase for
the uremic cells was significantly less than that for the normal
Table 2. Creatinine Equilibrium Partition Coefficients (K,) and RBC Membrane Permeabilities (k)for Uremic and Normal Cells as a
Function of Suspending Medium and Temperature
Type of
Cells
Suspending
Medium
Equilibrium Coefficient
Normal plasma
Uremic plasma
Normal plasma
Uremic plasma
0.72f 0.05(n = 10)
P8S
0.74? 0.09(n = 8)
PBS
-
K,
Permeability k
( 1O-s cm/s)
Significance'
37°C
Normal
Uremic
Normal
Normal
Normal
Uremic
0.72? 0.05(n = 10)
-
16.7? 3.8(n = 15)
7.2 k 2.0(n = 3)
16.7? 3.8(n = 15)
19.7f 2.8(n = 2)
8.7? 2.7(n = 12)
2.9? 0.9(n = 3)
P < ,001
NS
P < .01
30°C
Normal
Uremic
Normal
Normal
Normal
Uremic
Normal plasma
Uremic plasma
Normal plasma
Uremic plasma
PBS
PBS
0.74f 0.08(n = 9)
Normal plasma
Uremic plasma
Normal plasma
Uremic plasma
PBS
PBS
0.69f 0.08(n = 7)
0.74f 0.08(n = 9)
0.67f 0.02(n = 2)
-
7.3k 1.1 (n = 9)
5.6 +- 0.7 (n = 3)
7.3? 1.1 (n = 9)
6.7+- 1.1 (n = 3)
4.7k 1.4(n = 4)
2.0f0.1(n = 3)
P < .05
3.6f 0.6(n = 18)
2.8f 0.5(n = 4)
3.6 f 0.6(n = 18)
3.9 f 0.5(n = 5)
2.5f 0.6(n = 13)
1.8 f 0.4(n = 6)
P < .05
NS
P < .05
23°C
Normal
Uremic
Normal
Normal
Normal
Uremic
0.69 ? 0.08(n = 7)
0.71 f 0.04(n = 2)
-
NS
P < .05
* The statistical significance of the differences in permeabilities for comparative experiments (eg, normal Y uremic cells or normal v uremic plasma)
were determined using a Student's t-test.
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825
EFFECT OF UREMIA ON RBC MEMBRANE TRANSPORT
Table 3. Uric Acid Equilibrium Partition Coefficients (K,) and RBC Membrane Permeabilities (k) for Uremic and Normal Cells as a
Function of Suspending Medium and Temperature
Equilibrium Coefficient
K,
Cells
Suspending
Medium
37°C
Normal
Uremic
Normal
Normal
Normal
Uremic
Normal plasma
Uremic plasma
Normal plasma
Uremic plasma
PBS
PBS
0.52 t 0.07 (n = 4)
Normal plasma
Uremic plasma
Normal plasma
Uremic plasma
PBS
PBS
0.59 f 0.12 (n = 3)
Normal plasma
Uremic plasma
Normal plasma
Uremic plasma
PBS
PBS
0.73 f 0.07 (n = 10)
Type of
30°C
Normal
Uremic
Normal
Normal
Normal
Uremic
23°C
Normal
Uremic
Normal
Normal
Normal
Uremic
0.52 f 0.07 (n = 4)
-
0.52 f 0.03 (n = 7)
-
0.59 k 0.12 (n = 3)
-
0.63 f 0.04 (n = 4)
0.73 f 0.07 (n = 10)
-
0.72 f 0.01 (n = 3)
-
Permeability k
( 10-9 cm/s)
Significance'
12.2 f 2.7 (n = 10)
3.8 f 0.8 (n = 3)
12.2 f 2.7 (n = 10)
6.5 lr 1.8 (n = 4)
42.1 t 7.1 (n = 12)
41.2 f 8.5 (n = 3)
P < ,001
4.8fl.l(n=4)
2.9 f 0.9 (n = 2)
4.8?1.1(n=4)
3.3 t 0.8 (n = 2)
20.2 f 8.6 (n = 4)
19.9 lr 1.8 (n = 2)
P < .15
1.6 t 0.6 (n =
2.1 f 0.5 (n =
1.6 lr 0.6 (n =
2.4 k 0.5 (n =
1.9*0.8(n=
2.2 f 0.6 (n =
20)
4)
20)
6)
13)
5)
P < .01
NS
P<.2
NS
P c .15
P < .01
NS
The statistical significance of the differences in permeability for comparative experiments (eg, normal v uremic cells or normal v uremic plasma)
were determined using a Student's f-test.
cells for both solutes. This difference is also reflected in the
smaller activation energies for solute transport with the uremic blood, 11.8 k 3.2 versus 19.2 t 1.8 kcal/mol for creatinine and 7.4 t 0.4 versus 25.4 k 4.8 kcal/mol for uric acid,
with the activation energies evaluated directly from the slope
of the In k versus 1 / RT data in Fig 3. It is the large difference
in the activation energies, and thus in the temperature dependence of the permeability, that leads to the different effects
of uremia on the uric acid permeability at low (T = 23°C)
and high (T = 37°C) temperatures seen in Table 3.
The creatinine permeability for uremic cells in PBS was
significantly less than that for normal cells in PBS at all three
temperatures ( P < .01, P < .05, and P < .05 at 37, 30, and
23"C, respectively), while the creatinine permeability for
normal cells in normal and uremic plasma were essentially
identical at all three temperatures. These results are again
consistent with the hypothesis that the effect of uremia on
creatinine transport is due to an alteration in the uremic
cells. It is also worth noting that the creatinine permeability
of normal cells in PBS was consistently less than that for
normal cells in normal plasma with this difference increasing
from 30% at 23°C to 50% at 37°C.
The uric acid permeability for uremic cells in PBS was
essentially identical to that for normal cells in PBS at all three
temperatures. However, the permeability for normal cells in
uremic plasma was significantly less than that for normal
cells in normal plasma at 37°C ( P < .Ol), with this trend
reversed at 23°C (P< .01). This temperature effect was analogous to that seen previously for normal and uremic cells in
autologous plasma (Fig 3, bottom panel) and reflects the different activation energies for uric acid transport for normal
cells in normal and uremic plasma. These results again demonstrate that the effect of uremia on uric acid transport is
associated with some alteration of the uremic plasma and
not the uremic RBCs. The uric acid permeabilities for normal
cells at 23°C were similar in PBS and plasma, while at higher
temperatures the uric acid permeability in PBS was as much
as a factor of three greater than that in plasma. Similar effects
of suspending phase on the RBC permeability have been reported by Buzdygon' and are discussed in more detail subsequently.
DISCUSSION
Although previous studies of the effects of uremia on solute
transport across the RBC membrane are highly limited, there
have been several investigations of the permeability and
equilibrium partition coefficient of normal cells to solutes of
interest in hemodialysis, including both creatinine and uric
acid. Gary-Bobo and LindenbergI2 evaluated the creatinine
equilibrium partition coefficient for cells in Ringer's solution
as I(eq = 0.76 (independent of temperature), while Skalsky
et als reported Keq = 0.72 k 0.03 for cells in plasma at 37°C.
These values are in very good agreement with those obtained
in this study. The creatinine permeability for normal cells in
autologous plasma at 37°C reported by Skalsky et a1 (23.0
rfr 5.3 X
cm/s) was only slightly higher than that obtained
cm/s), with this slight difin this study (16.7 k 3.8 X
ference probably caused by the inherent variability between
donors.
The uric acid equilibrium partition coefficients obtained
in this study were also in good agreement with literature values
both at 37°C (K, = 0.52 +. 0.07 compared with 0.45 obtained
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
826
LANGSDORF AND ZYDNEY
100
L
a"
100
1
.
I
.
I
.
I
.
I
.
Uric Acid
activation
r
1
1
1.60
1
1.62
1.64
1.66
1.68
1.70
1.72
1
(mole/kcal)
RT
Fig 3. Effect of temperature on the RBC membrane permeability
for creatinine (top panel) and uric acid (bottom panel) for normal
and uremic cells in autologous plasma. Solid lines are linear regression fits to the data.
by Greger et all3) and at 23°C (Lq= 0.73 f 0.07 compared
with 0.72 f 0.1 1 measured by Benedict and Behrei4). This
reduction in
with increasing temperature has not been
discussed previously, but it may reflect the effect of temperature on the dissociation constant of uric acid in solution.
The pK, of uric acid decreases with increasing temperature,"
with the pK, decreasing by about 30% as the temperature
increases from 23°C to 37°C. This decrease in the pK, corresponds to about a 30% reduction in the concentration of
unionized uric acid, which may be directly associated with
the observed reduction in kqbecause uric acid is believed
to be transported across the RBC membrane only in its
unionized form."
The uric acid permeability for RBCs in plasma at 37°C
obtained in this study (12.2 f 2.7 X
cm/s) was in good
agreement with that reported previously by Greger et al(l5.0
X
cm/s). The uric acid permeabilities for normal cells
in PBS were slightly less than those obtained by Lassen"
using Krebs-Ringer-bicarbonate solutions at all three temperatures, which may simply reflect the slight differences in
RBCs suspended in PBS and in Krebs-Ringer-bicarbonate.
The lack of any concentration dependence for uric acid
transport in our experiments is not inconsistent with Lassen's
observations of carrier-mediated transport because all of the
data obtained in this study were at uric acid concentrations
well below the Michaelis-Menten constant (KM= 50 mg/dL)
reported for uric acid transport."
The only previous studies of the effect of uremia on solute
transport across the RBC membrane are those of Skalsky et
a15 for creatinine and of Fervenza et a16 for lysine. Data obtained in this investigation indicate that K, for creatinine is
unaffected by uremia, but the creatinine permeability for
uremic cells in uremic plasma at 37°C is about 55% less than
that obtained with normal cells. This is only slightly greater
than the 40% decrease in permeability reported by Skalsky
et al.' The relatively good agreement between these two results
suggests that Epo, which was used by the large majority of
patients examined in this study, has no significant effect on
the permeability of uremic RBCs to creatinine. The data obtained with uremic cells in PBS and with normal cells suspended in uremic plasma show that the reduction in creatinine permeability is directly associated with an alteration of
the RBC membrane, with the uremic plasma having no significant effect on creatinine transport.
The effect of uremia on uric acid transport was quite different from that on creatinine transport. At 37"C, the uric
acid permeability for uremic cells in uremic plasma was about
70% less than that for normal cells in normal plasma, but at
23°C the uremic permeability was actually slightly greater
than that for normal cells. This temperature effect was caused
by the large difference in the activation energies for uric acid
transport, with the activation energy for uremic cells in uremic
plasma more than a factor of three less than that for normal
cells in normal plasma. In addition, the effect of uremia on
the uric acid transport appears to be primarily associated
with the uremic plasma; the permeability for uremic cells in
PBS was essentially the same as that for normal cells in PBS,
while the permeability for normal cells in uremic plasma was
similar to that obtained with uremic cells in uremic plasma.
The large increase in RBC membrane permeability to uric
acid at 37°C on transfer of the uremic cells to PBS was similar
to the improvement in erythrocyte membrane function seen
by Perez-Fontan et a14 on transfer of uremic RBCs to Hanks'
medium .
The very different behavior seen with uric acid and creatinine most probably reflects the different transport mechanisms for these solutes. Creatinine transport across the RBC
membrane is primarily by passive diffusion through the lipid
bilayer, thus the RBC membrane composition would likely
be a dominant factor in determining the rate of creatinine
transport. The observed reduction in the creatinine permeability associated with uremia may be caused by the elevated cholesterol level3 and altered phospholipid content16
of the RBC membrane for cells obtained from uremic patients, with the uremic membrane offering a greater resistance to creatinine diffusion through the lipid bilayer. This
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
EFFECT
OF UREMIA ON RBC MEMBRANE TRANSPORT
increased transport resistance is also consistent with the increased rigidity of uremic RBCs, an effect that has also been
linked to this alteration in the membrane lipid c o m p o ~ i t i o n . ~
The data obtained in this study also indicate that the permeability can be restored to normal levels on incubation of
uremic cells in normal plasma. The exact mechanism by
which this occurs is not known, but this result is consistent
with the fairly rapid exchange of lipids and cholesterol between RBCs and plasma observed by Peuchant et a13 in
their studies of uremic RBCs. The lack of improvement in
creatinine transport seen on incubation of the RBCs in PBS,
which contained no cholesterol o r lipids, is also consistent
with this hypothesis.
The large effect of the uremic plasma on the uric acid
permeability suggests that there is some plasma constituent
which is responsible for the alteration of the RBC membrane
permeability to uric acid. This plasma constituent may be
related to the factor in uremic plasma that inhibits hexose
monophosphate shunt metabolism.” The inhibition of this
shunt metabolism is thought to cause an increase in RBC
membrane peroxidation” and a concomitant alteration in
the RBC deformability,” with the data on uric acid permeability from this study suggesting that it may also alter the
properties of the camer system involved in uric acid transport,
Further investigations would be required to determine the
exact nature of this plasma constituent and to better understand the biochemical and mechanical changes in both the
RBCs and plasma associated with uremia.
The very large effect of uremia on the RBC membrane
permeability can have important consequences for the
maintenance of physiologic concentrations of key metabolites for patients with end stage renal disease. For example,
the dramatic increase in the saturation half-time for uric
acid transport (from 54 minutes for normal cells t o 180
minutes for uremic cells) would cause a significant reduction in uric acid transport from RBCs to plasma during
hemodialysis, leading to a significant reduction in uric acid
clearance during dialysis. A similar, but somewhat smaller,
effect would be seen with creatinine. These long saturation
half-times would be particularly important in applications
of high-flux or high-efficiency dialysis, in which the typical
dialysis session is only about 120 minutes, significantly
shorter than the saturation half-time for uric acid for uremic blood cells. The large effects of uremia o n the RBC
membrane permeability also suggest that there may be alterations in intercellular transport rates throughout the
body, which could have important implications for the
transport and distribution of key metabolites and therapeutic agents in uremic patients. The restoration of the
creatinine permeability to normal levels on incubation of
uremic cells in normal plasma suggests that it might be
possible to improve transport of key metabolites by mod-
827
ification of the plasma (eg, by administration of a n appropriate chemical agent during dialysis).
ACKNOWLEDGMENT
The authors thank Dr Miles Sigler (Lankenau Hospital) and Dr
Anthony Zappacosta (Bryn Mawr Hospital) for donation of uremic
blood samples, and Marla Fosdick at the Blood Bank of Delaware
for assistance in obtaining normal blood.
REFERENCES
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in uremic hemodialyzed patients. Ann Intern Med 79:84 1, 1973
2. Rosenmund A, Binswanger U, Straub PW: Oxidative injury to
erythrocytes,cell rigidity and splenichemolysis in hemodialyzed uremic patients. Ann Intern Med 82:460, 1975
3. Peuchant E, Salles C, Vallot C, Wone C, Jensen R: Increase of
erythrocyte resistance to hemolysis and modification of membrane
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4. Perez-Fontan M, Zamorano A, Huarte E, de Castro M, Selgas
R: Effect of transfer of erythrocytesto a nonuremic medium on corpuscular parameters and red blood cell deformability in patients
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PhD Thesis, University of Delaware, 1990
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11. Lassen UV: Kinetics of uric acid transport in human erythrocytes. Biochim Biophys Acta 53557, 1961
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J Physiol (Paris) 52:106, 1960
13. Greger R, Lang F, PUISF, Deetjen P: Urate interaction with
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Chem 92:161, 1931
15. Finlayson B, Smith A: Stability of first dissociable proton of
uric acid. J Chem Eng Data 19:94, 1974
16. Bleiber R, Eggert W, Reichmann G, Kunze D: Erythrocyte
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117, 1980
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18. Carrel1 RW, Winterbourn CC, Rachmilewitz EA: Activated
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1993 81: 820-827
Effect of uremia on the membrane transport characteristics of red
blood cells
LJ Langsdorf and AL Zydney
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