J Appl Physiol 97: 1527-1534, 2004.
First published June 18, 2004; doi:10.1152/japplphysiol.00404.2004
8750-7587/04 $5.00
INNOVATIVE METHODOLOGY
Comparison of PEG-modified albumin and hemoglobin in extreme hemodilution in the rat
Robert M. Winslow,1,2
Jeff Lohman,1
Ashok Malavalli,1 and
Kim D. Vandegriff1
1Sangart, and 2Department of Bioengineering, University of California, San Diego, California 92121
Submitted 15 April 2004
; accepted in final form 29 May 2004
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ABSTRACT
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We have reported a new polyethylene glycol (PEG)-modified, hemoglobin-based O2 carrier (MP4) with novel properties, including a large molecular excluded volume and low PO2 necessary to obtain 50% O2 (
6 Torr). To evaluate the ability of MP4 to transport O2, we compared it with PEG-modified albumin (MPA) using the identical chemistry of attachment of PEG chains. The resulting solutions were well matched with respect to all physical properties except that MP4 is an O2 carrier, whereas MPA is not. An additional solution, 10% pentastarch, was matched with the PEG-modified proteins with regard to oncotic activity and viscosity but does not contain PEG. The model used to evaluate O2 transport was continuous exchange transfusion in the rat until the hematocrit was virtually unmeasureable. Objective end points included survival and the onset of anaerobic metabolism, signaled by acid-base derangement and accumulation of lactic acid. Continuous exchange transfusion of 2.5 blood volumes in rats (n = 5 in each treatment group) was carried out over 60 min, such that the final hematocrit was between 0 and 5% in all animals. Animals were observed for an additional 70 min, when survivors were killed. Overall survival for the MP4 animals was 100%; no animal that received either pentastarch or MPA survived. The hematocrit at which lactic acid began to rise was 14.8% in both pentastarch and MPA animals and 7.4% in the animals that received MP4. In all groups, the total hemoglobin was 5 g/dl at this point. We conclude that, despite its low PO2 necessary to obtain 50% O2, MP4 effectively substitutes for red blood cell hemoglobin in its ability to oxygenate tissues in extreme hemodilution.
polyethylene glycol-hemoglobin; polyethylene glycol-albumin; MP4; hemospan; blood substitute; hemodilution
OXYGEN-CARRYING RED CELL SUBSTITUTES have been developed in many forms, including human, animal, and recombinant hemoglobins (Hb), which may then be cross-linked, polymerized, or conjugated to various polymers including starches and polyethylene glycol (PEG) (24). The goals of such modifications include prolongation of the intravascular retention time, reduced toxicity, and assuring delivery of O2 to tissues. The most severe toxicity of cell-free Hb, i.e., renal failure caused by rapid glomerular filtration of unmodified Hb, appears to have been overcome by most modifications currently being studied, and intravascular retention times in the range of 24 to 48 h (half-life) appear to be adequate to significantly reduce the amount of blood transfused in most routine surgical procedures (7, 9, 10). The remaining goal, promotion of tissue oxygenation, is the most difficult to evaluate because measurements are indirect and invasive procedures cannot be used in many animal and most human models.
A major hurdle remaining in the development of Hb-based products is vasoconstriction, i.e., the tendency of Hb to cause narrowing of vessels in the arterial circulation. Vasoconstriction induced by low molecular weight Hb derivatives can lead to hypertension, reduced cardiac output, increased resistance, reduced tissue perfusion, and, paradoxically, reduced tissue oxygenation with consequent lactic acidosis (26). If used to resuscitate trauma patients with penetrating injuries, rapid restoration of blood pressure could also cause significant rebleeding (2). Clinical trials with an example of this type of product were discontinued because of excessive adverse reactions (14, 16). One unifying hypothesis to explain these clinical failures is that vasoconstriction may be responsible for a wide variety of toxic effects that are mediated by smooth muscle contraction, including hypertension and esophageal spasm (25).
The most popular theory to explain Hb-induced vasoconstriction is scavenging the endothelium-derived relaxing factor nitric oxide (NO) by Hb, which is known to have a very high NO affinity (6). With this working hypothesis, mutant Hbs have been expressed in bacterial and yeast systems that have reduced NO-heme affinity, and these mutants demonstrate a reduced tendency to produce vasoconstriction (5). Our group found, in contrast, that several examples of modified Hb had different effects on blood pressure in rats, even though the measured rates of reaction with NO were identical (13). Among these products was Hb modified with PEG, which had no significant hypertensive effect (26).
With the need for an explanation of Hb-induced vasoconstriction as an alternative to the NO hypothesis, we explored the possibility that this phenomenon could be a normal autoregulatory overreaction to excessive O2 supply to controlling arterioles (27). This theory was based on the ability of cell-free Hb to participate in "facilitated diffusion" (15) and suggested that the way to control vasoconstriction was to control the diffusive properties of Hb, which include molecular size, viscosity, and O2 affinity (19). This concept was tested in a simple artificial capillary system (11) in which PEG-modified Hb with a PO2 necessary to obtain 50% O2 (P50) of 12 Torr released O2 in a manner very similar to that of native red blood cells, whereas both unmodified Hb and Hb cross-linked between the 
chains demonstrated markedly increased release.
Based on these theoretical and experimental findings, we have developed a new PEG-modified Hb that employs novel sulfhydryl-maleimide conjugation chemistry (1). The modified Hb (MalPEG-Hb), as formulated at 4.2 g/dl in lactated Ringer USP, is designated MP4 (20). MP4 is produced from human Hb obtained from outdated human red blood cells. Its unique properties include high O2 affinity (P50 of 6 Torr), increased oncotic pressure (50 Torr), and viscosity (2.5 cPs), all of which are counter to conventional recommendations for the production of Hb-based O2 carriers (23). Because this product has such unusual properties, we wished to determine whether its beneficial effects are a result of O2 delivery by Hb or whether they are merely a reflection of oncotic volume expansion or some other property of PEG. In the present study, critical differences were compared in an animal model of extreme hemodilution. MP4 was compared with hydroxyethyl starch [pentastarch (PS), Pentaspan] and PEG-modified human albumin (MPA), oncotically matched to MP4. The primary objective end point was determination of the hematocrit at which lactic acid begins to accumulate (the critical hematocrit).
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METHODS
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Solutions.
Hemoglobin solutions were prepared and characterized as described previously (20). Briefly, outdated human packed red blood cells, obtained from the San Diego Blood Center, were washed with saline, lysed with distilled water, and diafiltered to achieve a Hb concentration of 4.2 g/dl in lactated Ringer solution (stroma-free hemoglobin). MalPEG-Hb was produced by the reaction of human Hb with 2-iminothiolane to introduce sulfhydryl groups at surface 
-amino groups (lysine), which were then conjugated to six strands per tetramer of maleimide-activated PEG (molecular mass 5 kDa; NOF, Tokyo, Japan) (1, 20). Preparation of MalPEG-albumin was done by the same procedure, substituting albumin for Hb. PS (Pentaspan) was a gift from B. Braun Medical (Irvine, CA). Human serum albumin (26 g/dl) was obtained from Alpha Therapeutics (Pasadena, CA).
Size-exclusion chromatography was performed using an AKTA Purifier-10 fast-performance liquid chromotography instrument (Pharmacia) using two Superose columns in series. Samples were eluted using PBS at pH 7.4 and a flow rate of 0.5 ml/min at room temperature.
The test solutions (see Table 1) were 1) PS, containing 10 g/dl PS; 2) MP4, containing 4.2 g/dl MalPEG-Hb in lactated Ringer USP; and 3) MPA, containing 5.0 g/dl MalPEG-albumin in lactated Ringer USP. Although the molecular mass of MalPEG-Hb is 95 kDa compared with 64 kDa for unmodified Hb, concentrations are expressed on heme basis. That is, concentrations are estimated from optical density and extinction coefficients for Hb, so that, gram for gram, MalPEG-Hb has the same O2 capacity as native Hb. O2 equilibrium curves were measured using the method described previously (21), and other physical characterizations, including viscosity and colloid osmotic pressure, were as previously reported (18). The concentration of MPA was chosen so as to match colloid osmotic pressure and viscosity with MP4 (see Table 1).
Animals.
The animal protocol was approved by the Sangart Institutional Animal Care and Use Committee and the University of California-San Diego animal welfare protocol committee. Male Sprague-Dawley rats were purchased from Charles River (Hollister, CA). After processing was completed, animals were permanently housed in the vivarium of the Bioengineering Department of the University of California-San Diego. After 2 days of quarantine, animals were acclimated to an immobilization device (Braintree Scientific, Braintree, MA) for several hours over several days before surgery. On the day of surgery, animals were anesthetized by intramuscular injection of a rodent cocktail containing a mixture of ketamine (40 mg/kg), acepromazine (0.75 mg/kg), and xylazine (3 mg/kg). Catheters made of polyetheylene tubing (Clay Adams PE-50 and PE-10) were implanted into both femoral arteries and one femoral vein. The catheters were externalized at the base of the tail and covered by a tail sheath for protection and future access. After closure of the surgical wounds, animals were returned to their cages and allowed to wake up and recover for 24 h before initiation of the experiment. Animals were given free access to food and water during recovery.
For the hemodynamic measurements, the femoral artery catheter was connected through a stopcock and a 23-gauge needle to a pressure transducer (UFI model 1050, Morro, CA), and arterial pressure was sampled continuously at 100 Hz using a MP100WSW data collection system (Biopac Systems, Goleta, CA). The data were stored in digital form for subsequent offline analysis. The arterial pressure signal was analyzed using a program written in FORTRAN. Heart rate for each beat was calculated as the reciprocal of the interval between successive pressure peaks. Systolic and diastolic pressures were the maximum and minimum pressures, respectively, and the mean arterial pressure (MAP) was diastolic + 1/3 (systolic – diastolic) pressure. Mean values of heart rate, systolic, diastolic, mean arterial pressures, and pulse pressure were averaged for each minute of data.
Blood gases, hematologic, and lactate measurements.
Arterial pH, PCO2, and PO2 were measured in a Bayer model 248 blood-gas analyzer using 100-µl heparinized samples of blood. Lactic acid was measured in femoral artery blood using a YSI lactate analyzer (Yellow Springs Institute, Yellow Springs, OH). Total CO2, standard bicarbonate (HCO
), and base excess (BE) were calculated from PCO2, pH, and Hb concentration using algorithms described previously (22). Plasma lactate was calculated as
where Lacplasma is plasma lactate, Lacblood is blood lactate, and Hct is hematocrit. Total Hb concentration was measured using a 
-Hb photometer (HemoCue, Mission Viejo, CA) using 50-µl samples of blood collected from the femoral artery. Plasma Hb was measured on a separate HemoCue instrument (a gift from HemoCue), which had been modified for an increased path length and allowed the measurement of very low Hb concentrations. Hematocrit was measured using 50-µl samples of arterial blood by microcentrifugation.
Exchange transfusion.
Fully conscious animals (n = 5 for each treatment group) were placed in Plexiglas restrainers. The arterial and venous cannulae were flushed with 200 and 100 µl, respectively, of heparinized saline (100 U/ml). The arterial and venous catheters were connected to an infusion pump (Labconco model 4262000, Kansas City, MO), and exchange-transfusion was carried out at a rate of 0.5 ml/min for 100 min. Thus the total volume of solution exchanged was 50 ml or 2.5 blood volumes. The peristaltic pump was operated so that blood was removed at exactly the same rate as test material was infused. Test solutions were warmed to 37°C in a water bath before infusion and kept warm during infusion by a heating pad. At the end of the 100-min exchange period, animals that survived were monitored for an additional 70 min before euthanasia. Blood samples (0.3 ml) were taken every 10 min for hematologic and blood-gas analysis.
Statistical and survival analysis.
Statistical analyses were done using Sigmaplot (SPSS). Unless otherwise noted, values are expressed as means ± SE. Differences between means were considered significant at P < 0.05. The time of death was determined to be the point at which pulse pressure (diastolic – systolic pressure) was <5 mmHg. Animals alive at 130 min after starting the exchange procedure were considered to be censored and were euthanized. For the survival analyses, data were analyzed according to Kaplan and Meier, and the probability of survival and its standard error were calculated. Analysis of significance of survival differences between groups was done using the Mantel-Cox test of significance.
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RESULTS
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Solutions.
The properties of the solutions used in the experiments are summarized in Table 1. MP4 and MPA were closely matched in oncotic pressure and viscosity. The concentrations given (4.2 and 5.0 g/dl) are the concentrations of protein in both cases. Because of the conjugation to PEG, the molecular mass of each is 95 kDa. We have expressed the concentration on protein basis so that O2 transport capacity can be compared directly with native Hb. The P50 of MP4 is 6 Torr.
The PEG modification reactions had similar effects on the properties of Hb and albumin (Table 1). In both cases, the viscosity increased from 1.0 to 2.2 cPs and the oncotic pressure from 15 to 49 Torr for Hb and from 20 to 50 Torr for albumin. Although the number of PEG strands per molecule was not measured directly for MPA, the reduction in peak retention time in size-exclusion chromatography from 50.2 to 43.0 min is similar to the change found for PEG modification of Hb (55.7–43.7 min). We thus conclude that, like MP4, MPA contains approximately six strands of 5,000-kDa PEG (20).
Survival.
The end of the survival period was defined as a pulse pressure (systolic – diastolic) of <5 mmHg (Fig. 1, top). This allowed an unambiguous time of death for the Kaplan-Meier analysis of survival. (Fig. 1, bottom). All of the animals that received MP4 survived for the entire 130-min period of exchange transfusion, whereas none of the PS or MPA animals did. There was no difference in survival for the latter two groups, suggesting that the presence of PEG did not confer any protection. Because the oncotic pressures of all three solutions were similar (Table 1), we conclude that prolonged survival for the MP4 animals is not a result of greater volume expansion.
Hematology.
The hematocrit fell exponentially (Fig. 2, top), reaching virtually undetectable levels at 100 min after the exchange process was started in the MP4 animals. The hematocrit level in the PS and MPA animals did not fall as low as it did in the MP4 animals because animals in these groups did not survive as long. The hematocrit fall was slightly greater for the MP4 compared with MPA or PS animals, suggesting greater volume expansion in those animals. In the animals that received either PS or MPA, total Hb declined to 2 g/dl at the end of the exchange procedure, whereas in the animals that received either MP4 total Hb was significantly higher (Fig. 2, bottom). The total Hb was a linear function of hematocrit in all animals (Fig. 3, top), as was plasma Hb in the MP4 animals (Fig. 3, bottom).
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Fig. 2. Hematologic response to continuous exchange transfusion. Hematocrit reduction is more profound in animals that received solutions with relatively higher colloid osmostic pressure (COP) (top), but total hemoglobin is higher in animals that received MP4 (bottom). Values are means ± SE.
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Fig. 3. Total hemoglobin (top) and plasma hemoglobin (bottom) as a function of hematocrit during exchange transfusion. Values are means ± SE.
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Hemodynamics.
Systolic and diastolic blood pressures are plotted on a minute-by-minute basis in Fig. 4. In the figure, the start of the exchange procedure is indicated by an arrow, showing a slight rise in both diastolic and systolic pressures when the procedure begins. As reflected in Fig. 1, the pulse pressure diminishes in both the PS and MPA animals but is maintained in the MP4 animals. It appears that, in both the PS and MPA animals, there is the beginning of hemodynamic recovery at 90 min, but it is short lived and ineffective.
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Fig. 4. Systolic (closed symbols) and diastolic (open symbols) blood pressures for each of the animal groups. A: PS. B: MPA. C: MP4. The exchange procedure begins at the arrow in each case.
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After initiation of the exchange procedure, the MAP of the animals that received MP4 increased slightly, but there were no significant between-group differences until 40 min of exchange (Fig. 5, top). At that point, MAP in both the PS and MPA groups began to fall. Although the MAP tended to drift downward for the duration of the experiment in the MP4 animals, it remained >75 mmHg at the time of euthanasia.
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Fig. 5. Hemodynamic response to continuous exchange transfusion. Top: mean arterial pressure (MAP) increases slightly after initiation of the exchange but is better maintained for the duration of the experiment in the animals that received MP4 compared with those that received non-O2 carriers (MPA, PS). Bottom: heart rate response is more immediate in the PS and MPA animals. Values are means ± SE.
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The heart rate rose almost immediately in the animals that received the non-O2 carriers (MPA, PS) and only somewhat later in the animals that received MP4 (Fig. 5, bottom). The heart rate in the PS and MPA animals fell sharply at 40 min, even though the first death in these two groups did not occur until 60 min. Maintenance of heart rate was best in the animals that received MP4. Shock index (heart rate/systolic blood pressure, not shown) is a general index of shock stress. Significant between-group differences are seen as early as 20 min after initiation of the exchange transfusion, where the values for animals that received MP4 are lower than those in the animals that received either PS or MPA.
Acid-base and blood gas regulation.
As the exchange transfusion progressed, animals hyperventilated, as reflected in rising arterial PO2 (PaO2; Fig. 6, top) and falling arterial PCO2 (PaCO2; Fig. 6, bottom). This effect appears to be least pronounced for the animals that received MP4. At the last time point at which all animals are alive (50 min), PaO2 is significantly lower and PaCO2 significantly higher in the MP4 animals compared with all other groups, and there are no significant differences among the PS and MPA groups. At that point, total Hb (Fig. 2, bottom) is highest in the MP4 animals. These findings suggest the stimulus to hyperventilate was less in the MP4 animals.
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Fig. 6. Ventilatory response to hemodilution. Arterial PO2 (PaO2; top) and arterial PCO2 (PaCO2; bottom) changes are mirror images. Values are means ± SE.
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Acid-base regulation deteriorates significantly after 50 min in the animals that received PS and MPA, as reflected in both pH and BE (Fig. 7). Acid-base control is significantly better in the MP4 animals compared with the PS and MPA groups. It is interesting to note that the MP4 animals demonstrate the least hyperventilation (Fig. 6), which, in the context of better acid-base control, indicates better overall homeostasis in this group of animals compared with all of the other groups.
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Fig. 7. Acid-base response to hemodilution. The pH (top) and base excess (bottom) are shown during continuous exchange. Values are means ± SE.
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Critical hematocrit.
The "critical" hematocrit is indicated by many of the variables measured in these experiments, all of which demonstrate a change at 40 min after start of the exchange transfusion. This is true of MAP (Fig. 5, top), heart rate, PaCO2 (Fig. 6, bottom), pH (Fig. 7, top), and BE (Fig. 7, bottom). With the use of lactic acid as an indicator of global O2 sufficiency, the critical hematocrit in animals hemodiluted with either PS or MPA is 15% (Fig. 8, top). In contrast, the animals that received the O2 carrier demonstrated a lowered critical hematocrit, by 10 percentage points, to 5%. Viewed as a function of total Hb concentration (Fig. 8, bottom), the critical point in O2 delivery is reached at 5 g/dl, regardless whether the Hb is located in red blood cells or plasma. This equivalence is evidence that the Hb component of MP4 (MalPEG-Hb) is as effective at O2 delivery as red cell Hb.
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Fig. 8. "Critical" hematocrit and hemoglobin. Lactic acid accumulation during exchange transfusion is shown as a function of hematocrit (top) or total hemoglobin (bottom). Values are means ± SE.
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Submaximal hemodilution.
At 40 min of exchange transfusion, all animals in all groups are still alive, but some differences are clearly apparent (Table 2). Among these, the hematocrit and total Hb are significantly lower in the MP4 animals compared with PS and MPA, whereas plasma Hb is 2.38 g/dl in these animals. Although not significant, the lactic acid has begun to rise in both the MPA and PS animals, whereas it remains at baseline in the MP4 animals. PaCO2 is significantly lower and PaO2 is higher in the MPA compared with MP4 animals, indicating a greater degree of hyperventilation in the MPA animals. BE is significantly higher in the MP4 compared with PS animals, and MAP in the MP4 animals is significantly higher than that in either of the control groups. Finally, the shock index (heart rate/MAP) is significantly lower in the MP4 compared with the PS or MPA animals. Taken together, these data clearly show that, even at submaximal hemodilution, delivery of O2 to tissues is more adequate in the MP4 compared with the PS or MPA animals.
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DISCUSSION
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The purpose of the present experiments was to determine whether a new formulation of PEG-modified human Hb (MalPEG-Hb) could serve as a surrogate for O2 transport by red blood cells. MPA was prepared using the same reaction chemistry as is used to prepare MalPEG-Hb, and it was formulated so that the solution properties (oncotic pressure and viscosity) would be identical (50 Torr and 2.2 cPs, respectively). Thus study of these two modified proteins allows conclusions to be drawn about one property: the capacity to transport O2. The main conclusion of the study is that the beneficial effect of MP4 in supporting life at extremely low hematocrit is a result of its ability to delivery O2 to tissues, not its oncotic pressure or viscosity.
MalPEG-Hb is currently formulated for human clinical development as 4.2 g/dl in lactated Ringer USP (MP4) (20). Two concerns are specifically addressed in this study: 1) that the P50 of MP4 is so low (6 Torr) as to prevent release of O2 to tissues, and 2) that the oncotic pressure of MP4 ( 50 Torr) is such that its efficacy is due solely to its ability to rapidly and effectively expand the blood volume. To satisfy both of these concerns, we compared the effects of MP4 with MPA prepared by using the identical chemistry so that the molecules were comparable in every regard except that MPA does not bind O2. The protocol involved a continuous exchange transfusion, carried out until the hematocrit was nearly unmeasurable. In the case of MP4 animals, essentially all O2 was supplied by the plasma Hb. The study was not designed for long-term effects or survival.
We found that animals exchange transfused with MP4, but not MPA or PS, survived the entire exchange period with no deaths. All animals in the MPA and PS groups died within the period of the experiments. The study thus confirms that MP4 is able to oxygenate tissues and that the effect is not due to any alternate property of PEG, because MPA was not effective. Furthermore, the study shows that, on a gram-for-gram basis, MalPEG-Hb is as effective in transporting O2 as red cell Hb, despite its low P50. This latter conclusion is in agreement with our laboratory's previous findings (11) that PEG-Hb and human red blood cells release O2 in approximately the same manner in an artificial capillary exposed to pure N2.
It appears that, given enough time, even the animals exchange transfused with MP4 might not have survived: lactic acid was increasing and BE was decreasing at the end of the observation period. Thus a plasma Hb level of 2–2.5 g/dl may not be sufficient to support life indefinitely in rats. However, due to the elevated oncotic pressure of MP4, it may not be possible to achieve a much higher plasma Hb concentration than this, and it might not be necessary in the majority of clinical cases, whereas hematocrit rarely reaches the low levels of these experiments.
Similar concerns were raised in regard to a different product, PEG-modified bovine Hb developed as a tumor radiation enhancer by Enzon. In response, Enzon conducted a comparison of PEG-bovine Hb, PEGylated human serum albumin, oxidized PEGBvHb, and PEGBvHb liganded to CO (3). These authors performed an 85% hematocrit reduction by hemodilution with test material and showed clearly that PEG-Hb led to superior survival, even 2 wk postdosing. In this case, sufficient red blood cells remained after exchange transfusion to ensure adequate tissue supply of O2.
An additional value of the present experiments is that they show a distinct lowering of the critical hematocrit compared with either PS or MPA. At a plasma Hb of 2 g/dl, MP4 is able to reduce the critical hematocrit from 15 to 7% in this model.
In a theoretical model, Hoeft et al. (8) found that, to maintain normal O2 consumption in the heart, the critical hematocrit in the coronary circulation is 14% and the Hb concentration at this hematocrit is 4.7 g/dl, which are values very close to our findings for PS and MPA. Hyperoxia, raising the PaO2 to 400 Torr, was found to lower the hematocrit to 12%. However, increasing the O2 consumption by a factor of 3, as might be seen in stress, raised the critical hematocrit to 21%. In the intestinal microcirculation, Van Bommel et al. (17) found that, at a hematocrit of 16%, tissue oxygenation became supply dependent, suggesting this level for the critical hematocrit of the gastrointestinal tract. Thus it would be expected that the actual value of the critical hematocrit might vary by organ, by patient, and in different age groups (4).
Messmer (12) has recommended that perioperative hemodilution should be performed to 20–25%. The present results suggest that MP4 could be effective for moderate hemodilution. However, its relatively low O2 capacity, compared with blood, suggests that its maximal potential would be realized when some red cells are present. Additional experiments need to be performed to model this clinical scenario.
Our experiments leave open the question of the optimal balance between O2-carrying capacity (Hb concentration) and plasma expansion capability (oncotic pressure). These parameters may be of critical importance when a solution is designed for use in patients with cardiovascular disease, for example, who might have a poor cardiac output response to volume load as opposed to patients with a healthier response. Clinical development of such products should be thus careful and prudent, keeping in mind the needs and limitations of individual patient groups.
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GRANTS
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This work was supported, in part, by National Heart, Lung, and Blood Institute Grants RO1 HL-64579, R43 HL-64996, R44 HL-62818, R24 HL-64395, R01 HL-40696, R01 HL-62354, and R01 HL-62318.
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DISCLOSURES
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R. M. Winslow, K. D. Vandegriff, J. Lohman, and A. Malavalli are employees of Sangart and hold stock options in the company. R. M. Winslow is President, Chief Executive Officer, and Board Chairman of Sangart.
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ACKNOWLEDGMENTS
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We are especially grateful to the San Diego Blood Center for provision of blood used in the manufacture of MalPEG-Hb.
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FOOTNOTES
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Address for reprint requests and other correspondence: R. M. Winslow, Sangart, 11189 Sorrento Valley Rd., Suite 104, San Diego, CA 92121 (E-mail: rwinslow{at}sangart.com).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Copyright © 2004 by the American Physiological Society.
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ABSTRACT
METHODS
), polyethylene glycol (PEG)-modified hemoglobin (MP4; 
), and PEG-modified albumin (MPA; 
). Values are means ± SE.