In-Vitro Myoglobin Clearance by a Novel Sorbent System

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 (<780 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 by > 90% 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 glomerular filter and becomes inspissated in the renal tubules [1]. While prophylactic hemodialysis or hemofiltration with high-permeability dialysis membranes can remove substantial 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 µl 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 human serum from three different donors, to give initial myoglobin concentrations of 55,000–75,000 ng/ml. This solution was perfused through an X-Sorb column identical 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 µl 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 decreased 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.

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

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.

Figure 1.  Reduction in Myoglobin (%) in serum by X-Sorb.

Table 2. Percent Reduction of Myoglobin Content in Serum by X-Sorb® Perfusion

Time of Perfusion (minutes)	% Decrease in Myoglobin Concentration (±Standard Deviation)
15	35.9±3.3
30	48.3±1.6
45	60.8±2.8
60	65.7±1.8
90	76.1±2.9
120	80.4±1.8
180	87.6±0.5
240	91.9±0.3

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 concentration 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 circulating myoglobin from the blood [2], [3]. However, some studies have found that dialysis or hemoperfusion even 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 hemoperfusion, might be useful to lower plasma myoglobin below the critical point and prevent this complication of acute rhabdomyolysis.