In-Vitro Myoglobin Clearance by a Novel Sorbent System

Viktoriya I. Kuntsevich, Donald A. Feinfeld, Pat F. Audia, Wendell Young,

Vincent Capponi, Marianna Markella and James F. Winchester
Division of Nephrology and Hypertension, Department of Medicine, Beth Israel Medical Center, New York, New York, USA
Abstract: Rhabdomyolysis may lead to acute kidney injury following deposition of myoglobin in renal tubules. Although high-flux
dialysis membranes may remove a substantial amount of myoglobin from plasma, this may still not be sufficient to prevent renal
damage. We tested a new polymer sorbent, X-Sorb

, in vitro to determine its potential to clear myoglobin from solutions. Normal saline
or human serum in which myoglobin was dissolved was perfused by a peristaltic pump through a column packed with the sorbent. After
a 4-hour perfusion, the myoglobin level in normal saline fell from 200,000 ng/ml to virtually undetectable ( B780 ng/ml). Perfusion
through the sorbent was then found to lower concentrations of dissolved myoglobin in 3 different 110-ml samples of human serum
consistently by90% over 4 hours. X-Sorb appears to be an effective sorbent for myoglobin and warrants a trial in vivo to determine
whether it is equally effective and safe.
INTRODUCTION
Rhabdomyolysis can result in acute kidney injury from
myoglobinuria when the myoglobin released into the
blood from damaged muscle passes through the glomer-
ular filter and becomes inspissated in the renal tubules [1].
While prophylactic hemodialysis or hemofiltration with
high-permeability dialysis membranes can remove sub-
stantial amounts of myoglobin from the blood, thus far
even the best myoglobin clearances have failed to
eliminate this protein entirely from plasma [2,3]. The use
of sorbents may improve the removal of large molecules
from the circulation [4]. We have tested in vitro a new
polymer sorbent (X-Sorb

, MedaSorb Technologies,
Monmouth Junction, NJ), which appears to have the
potential to clear myoglobin effectively from the blood.
MATERIALS AND METHODS
All tests were performed in vitro as simulated dynamic
experiments mimicking hemoperfusion at a ratio of 1ml of
wet X-Sorb polymer to 10 ml of either normal saline (0.9%
NaCl, Injection USP, B Braun Melsungen, Germany), or
human serum (Lampire Biological Laboratories, Inc). The
circuit consisted of the 10 ml column (Supelco, Bellefort,
PA) packed with wet polymer (X-Sorb), tubing, a reservoir
containing either normal saline or serum, with a magnetic
stirrer, and propelled by a peristaltic pump.
Normal Saline Solution Experiment
Myoglobin (Equine, M0630, Sigma-Aldrich) with an
initial concentration of 200,000 ng/ml in 0.9% NaCl
was pumped through the X-sorb column for one hour with
flow rate about 13 ml/min, modeling a flow rate of 400ml/
min for a 300ml device. Aliquots of 80 ml were collected
at 0, 15, 30, 45 and 60 min. The concentration of
myoglobin was calculated by direct measurement of light
absorbance at 410 nm (TIDAS I System, World Precision
Instruments). A calibration curve was created using
equine myoglobin solutions of known concentrations.
Human Serum Experiments
Three dynamic experiments were performed over 4 hours,
mimicking hemoperfusion. Human myoglobin (Biodesign
International, Saco, ME) was dissolved in 110 ml of
This study was presented in part at the annual meeting of the American Society of Nephrology, November 2006, and published as
an abstract, J. Am. Soc. Nephrol., 17, 720A (2006).
Wendell Young is a Senior Scientist and Vincent J. Capponi is Chief Operating Officer of MedaSorb Technologies. James F.
Winchester is a consultant to MedaSorb Technologies.
Address correspondence to James Winchester, MD, Division of Nephrology & Hypertension, Beth Israel Medical Center, 350 East
17
th
Street, New York, NY 10003, USA. E-mail: [email protected]
Artificial Cells, Blood Substitutes, and Biotechnology, 37: 4547
Copyright # 2009 Informa UK Ltd.
ISSN: 1073-1199 print / 1532-4184 online
DOI: 10.1080/10731190802664379
45
human serum from three different donors, to give initial
myoglobin concentrations of 55,00075,000 ng/ml. This
solution was perfused through an X-Sorb column iden-
tical to that used in the saline experiments, at a flow rate
of 13 ml/min, again modeling a 400 ml/min flow for a
300 ml device. Serum samples of 80 ml were collected at
the following time points: 0, 15, 30, 45, 60, 90, 120, 180
and 240 min. Concentration of myoglobin was estimated
by Enzyme Immunoassay (Life Diagnostics, Inc., West
Chester, PA) immediately after each experiment.
RESULTS
Normal Saline Solution Experiments
We found substantial removal of myoglobin from normal
saline solution (Table 1). A 60-minute perfusion de-
creased myoglobin concentration in saline from 200,000
ng/ml to less than 780 ng/ml, the lower limit for direct UV
detection of myoglobin solution.
Human Serum Experiments
After 4 hours of perfusion of serum though the X-Sorb
column the level of myoglobin decreased from 55174 ng/
ml, 55918 ng/ml and 72110 ng/ml down to 4343 ng/ml,
4451 ng/ml and 6110 ng/ml, respectively. The mean
percentage reduction in myoglobin in all three serum
samples at any given time point was remarkably similar
(Figure 1). The mean percentage reductions in myoglobin
and standard deviations are given in Table 2.
DISCUSSION
Large amounts of myoglobin in the blood can cause renal
injury by provoking constriction of renal vessels, forming
obstructing casts in the lumina of renal tubules, and
initiating interstitial inflammation [5]. A small case series
suggests that following rhabdomyolysis, the actual con-
centration of myoglobin in the urine, which correlates
with the blood level, may be an important factor in
determining whether kidney injury will occur [6]. While
early and vigorous intravenous infusion of isotonic fluids
may help prevent myoglobinuric renal failure [7,8], a
means of clearing myoglobin from plasma rapidly might
also decrease the risk of acute kidney injury.
Hemodialysis with membranes is not effective in
lowering plasma myoglobin levels [9]. Newer, high-flux
membranes are much more effective in clearing circulat-
ing myoglobin from the blood [2,3]. However, some
studies have found that dialysis or hemoperfusion even
Table 1. Concentration of Myoglobin in Normal Saline
Perfused Through X-Sorb

Time Myoglobin concentration (ng/ml)

0 min 200,000
15 min 24,860
30 min 1,814
45 min Undetectable
0
10
20
30
40
50
60
70
80
90
100
0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255
Time (minutes)
%

M
y
o
g
l
o
b
i
n

R
e
d
u
c
t
i
o
n
Series1
Figure 1. Reduction in Myoglobin (%) in serum by X-Sorb.
46 V. I. Kuntsevich et al.
with the high-permeability membranes does not always
cause a substantial fall in myoglobin levels [10,11].
Additionally, in those cases of effective clearance of
myoglobin from blood by high- or ultra-high-flux
membranes, the final plasma myoglobin levels were not
reduced below 16,000 ng/ml, which may still be high
enough to affect renal function [3,12]. X-Sorb

appears
to be an effective sorbent for myoglobin and warrants a
trial in vivo to determine whether it is equally effective
and safe. Such a sorbent, which could be added as a
cartridge in series with high-flux dialysis or hemoperfu-
sion, might be useful to lower plasma myoglobin below
the critical point and prevent this complication of acute
rhabdomyolysis.
REFERENCES
1. Zager, R.A. (1996). Kidney Int. 49:314326.
2. Maduell, F., Navarro, V., Cruz, M.C., Torregrosa, E.,
Garcia, D., Simon, V., Ferraro, J.A. (2002). Am. J. Kidney
Dis. 40:582589.
3. Naka, T., Jones, D., Baldwin, I., Fealy, N., Bates, S., Goehl,
H., Morgera, S., Neumeyer, H.H., Bellomo, R. (2005). Crit.
Care 9:R90R95.
4. Winchester, J.F., Ronco, C., Brady, J.A., Cowgill, L.D.,
Salsberg, J., Yousha, E., Choquette, M., Albright, R.,
Clemmer, J., Davankov, V., Tsyurupa, M., Pavlova, L.,
Pavlov, M., Cohen, G., Horl, W., Gotch, F., Levin, N.
(2002). Blood Purif. 20:8186.
5. Zager, R.A. (1992). Ren. Fail. 14:341344.
6. Feinfeld, D.A., Cheng, J.T., Beysolow, T.D., Briscoe, A.M.
(1992). Clin. Nephrol., 38:193l95.
7. Ron, D., Taitelman, U., Michaelson, M., Bar-Joseph, G.,
Bursztein, S., Better, O.S. (1984). Arch. Intern. Med.
144:277280.
8. Dubrow, A., Flamenbaum, W. (1988). Acute Renal Failure,
Brenner, B.M., Lazarus, J.M. (Eds.), Churchill Livingstone,
New York, 2nd ed, Chap. 10, pp. 279293.
9. Hart, P.M., Feinfeld, D.A., Briscoe, A.M., Nurse, H.M.,
Hotchkiss, J.L., Thomson, G.E. (1982). Clin. Nephrol.,
l8:141143.
10. Stefanovic, V., Bogicevic, M., Mitic, M. (1993). Int. J.
Artif. Organs 16:659661.
11. Shigemoto, T., Rinka, H., Matsuo, Y., Kaji, A., Tsukioka,
K., Ukai, T., Shimaoka, H. (1997). Ren. Fail. 19:711719.
12. Amyot, S.L., Leblanc, M., Thibeault, Y., Geadah, D.,
Cardinal, J. (1999). Intens. Care Med. 25:11691172.
Table 2. Percent Reduction of Myoglobin Content in Serum by
X-Sorb

Perfusion
Time of Perfusion
(minutes)
% Decrease in Myoglobin Concentration
(9Standard Deviation)
15 35.993.3
30 48.391.6
45 60.892.8
60 65.791.8
90 76.192.9
120 80.491.8
180 87.690.5
240 91.990.3
This paper was first published online on iFirst on 8 January 2009.
In-Vitro Myoglobin Clearance by a Novel Sorbent System 47