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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Diaspirin crosslinked hemoglobin (DCLHb) is a cell-free hemoglobin derived from human erythrocytes. DCLHb has been shown to improve blood flow to vital organs in healthy and septic animals. In this study, we determined the efficacy of DCLHb by comparing its effect on systemic O2 uptake to freshly stored and aged red blood cells (RBCs) in septic rats. Twenty-four hours after induction of sepsis by cecal ligation and perforation, O2 supply dependency was created by isovolemic hemodilution with rat plasma. In O2 supply dependency, rats were randomized to receive an exchange transfusion of 7.5 ml "fresh" RBCs (stored < 6 d; Hct: 70%), "fresh" diluted RBCs (stored < 6 d; Hct: 30%), "old" RBCs (stored 28 to 35 d; Hct: 70%), or DCLHb (Hb: 100 g/L). We found, that survival following O2 supply dependency and transfusion with old RBCs was poor (33% versus 91.7% in the other groups; p < 0.01), precluding further analysis of post-transfusion data from this group. Systemic O2 uptake increased in all remaining groups (p < 0.001), while systemic O2 delivery increased with "fresh" RBCs (p < 0.0001) and "fresh" diluted RBCs (p < 0.05) but not with DCLHb. Systemic O2 extraction increased with DCLHb as compared to baseline (p < 0.05) and to the other groups (p < 0.0001). Improved tissue oxygenation was associated with an increase in blood pressure and a fall in arterial lactate in all groups. We conclude that transfusion of DCLHb or "fresh" RBCs was efficacious at increasing systemic O2 uptake in O2 supply-dependent, septic rats.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Diaspirin crosslinked hemoglobin (DCLHb) is a cell-free hemoglobin solution characterized by a high O2-carrying capacity and a slightly right-shifted O2 dissociation curve as compared to human blood (1, 2). In hemorrhage models, DCLHb infusion increased blood pressure (3, 4), restored base deficit as rapidly as whole blood at half the volume (3), and improved subcutaneous tissue oxygenation (5). DCLHb also increases cardiac output and vasoconstrictor tone, presumably related mostly to NO-binding properties (6). Although numerous studies have characterized the effects of DCLHb on the circulation, its efficacy with regard to modifying tissue O2 uptake has not been explicitly demonstrated.
A fundamental objective of transfusing red blood cells (RBCs), and blood substitutes is to increase tissue O2 availability. Previous work has concluded that transfused RBCs are efficacious, with the demonstration of acceptable levels of 2,3-DPG and p50 in preserved cells and > 80% in vivo survival 24 h after transfusion (11, 12). This approach implicitly assumes that tissue oxygenation acutely increases post-transfusion when storage preservation techniques result in RBCs with acceptable biochemical function and viability (11, 12). A more direct approach to evaluate the efficacy of stored RBCs and blood substitutes is the demonstration of a temporally related increase in tissue O2 uptake. Thus, if transfusion is carried out when the tissues are in a state of O2 need, for example, when systemic O2 uptake is supply dependent, direct evidence for efficacy would be an increase in O2 uptake post-transfusion. Demonstrating a temporally related fall in elevated arterial lactate levels may also indicate efficacy of an O2-carrying agent since acute changes in arterial lactate have been used to represent changes in the balance between tissue O2 need and availability (13, 14).
Sepsis is a clinical syndrome distinguished by systemic inflammation and widespread tissue injury. This syndrome is also characterized by significant abnormalities at all levels of the circulation; for example, circulatory failure and an O2- extraction deficit (15, 16) are principle abnormalities in sepsis. As DCLHb infusion elicited a pressor response and improved regional blood flow to select tissues in septic rats (17), such data points to the possibility that DCLHb may be used in clinical protocols that include pressor and colloid fluid therapy in the treatment of sepsis. We therefore designed an experiment to characterize the efficacy of DCLHb in a clinically relevant small animal model of sepsis. We hypothesized that isovolemic exchange transfusion of DCLHb in rats made septic by cecal ligation and perforation, and in a state of acute O2 need, would be accompanied by an increase in O2 uptake. We found that DCLHb was efficacious when compared to fresh, stored RBCs and that this benefit was mediated at least in part by an increase in O2 extraction.
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INTRODUCTION
METHODS
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DISCUSSION
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Experimental Model
Forty-eight male Sprague-Dawley rats, weighing 320 to 380 g, were used after a 1-wk acclimatization period in our laboratory. Anesthesia was induced and maintained by halothane inhalation. Catheters were advanced into the superior vena cava (via the right external jugular vein) and left carotid artery. A thermodilution cardiac output probe (IT-21 thermocouple; Physiotemp Instruments, Clifton, NJ) was then positioned in the aortic arch via the carotid artery. After cannulation, rats underwent laparotomy and cecal ligation and perforation (two perforations with an 18-gauge needle) to establish sepsis. Postoperatively, rats were placed in individual cages where hemodynamic monitoring could be performed in the awake state, and fluid resuscitation with 0.9% saline (2 ml/100 g/h, intravenously) was started. Water and rat chow were available ad libitum. The carotid line was continuously flushed with heparin solution (45 IU/h) to maintain patency, and fentanyl (2 µg/100 g/h, intravenously) was added for analgesia.
Experimental Protocol
Twenty-four hours after surgery, rats were placed in a metabolic cage and the arterial and venous lines were connected to withdrawal and infusion pumps, respectively. After a 30-min acclimatization period, baseline mean arterial blood pressure, cardiac output, central venous pressure, and systemic O2 uptake were measured. Arterial blood (0.7 ml) was withdrawn for determination of hemoglobin concentration, arterial O2 saturation, and lactate concentration. Isovolemic hemodilution at a rate of 6 ml/h was then carried out to place the animal in O2 supply dependency. Systemic O2 uptake was monitored semi-continuously, and measurements of mean arterial pressure and central venous pressure were repeated after every 2 ml of isovolemic hemodilution. Simultaneously, blood samples for hemoglobin concentration, arterial O2 saturation, and lactate were obtained. At all times, shed blood was replaced by identical volumes of warmed plasma. After O2 supply dependency was created, the rats were then randomized to receive an isovolemic exchange transfusion with 7.5 ml of either: (1) "fresh" packed RBCs (Hct: 70 to 75%; positive control), (2) "fresh" diluted packed RBCs (Hct: 30%; hemoglobin-matched control), (3) "old" stored packed RBCs (Hct: 70 to 75%), or (4) diaspirin crosslinked hemoglobin (DCLHb; total dose of 750 mg) at a rate of 6 ml/h. Randomization and preparation of blood products for transfusion were performed in a separate laboratory room by a colleague who was not involved in this experiment. The investigator was, therefore, blinded to the randomization until the decision to commence transfusion was made. Less than 15 min before transfusion, RBCs or DCLHb were drawn into a syringe and warmed to body temperature. Hematocrit was measured when RBCs were prepared to certify an adequate RBC content, while hemoglobin was determined in DCLHb transfusions. Directly before transfusion, RBCs and DCLHb were filtered through a 40-µm transfusion filter (Alpha Micron-40; Alpha Therapeutic Corp., Los Angeles, CA). After transfusion, a 30-min stabilization period was allowed before complete measurements were repeated as described for baseline. After completion of the measurements, the rats were euthanized. Six rats treated with fresh diluted RBCs and five rats of the DCLHb group underwent postmortem examination. In these animals, bronchoalveolar lavage was performed on the left lung to determine the presence or absence of intra-alveolar hemoglobin. The right lung was removed to determine wet/dry ratio. The protocol of this study was approved by the Council on Animal Care of the University of Western Ontario (London, Canada).
Isovolemic Hemodilution and O2 Supply Dependency
For transfusion, rat plasma warmed to body temperature was filtered through a 40-µm transfusion filter. Using syringe pumps (Razel Scientific Instruments Inc., Stamford, CT) set at a rate of 6 ml/h, blood was withdrawn via the arterial line and plasma was infused via the jugular line. This way, O2 delivery was lowered in a stepwise manner to decrease it beyond critical O2 delivery. Below the point of critical O2 delivery, systemic O2 uptake becomes supply-limited (18, 19) and, therefore, decreases if systemic O2 delivery is further reduced. In the present study, O2 supply dependency was assumed after a > 30% decrease in systemic O2 uptake from baseline. We previously standardized this procedure of creating whole body O2 supply dependency and demonstrated its usefulness to study the efficacy of RBC preparations (20). To confirm supply dependency, an increase in lactate of > 250% from baseline was also required. In previous studies with this model, we noted that treatment of septic rats hemodiluted to O2 supply dependency by infusion of placebo (normal saline) alone was followed by death within 30 min in four of five studied animals (21).
Collection and Storage of Plasma and RBCs
To obtain rat plasma and RBCs, two donor rats were bled for each of
the 48 rats that underwent the experimental procedure. Donor rats
were anesthetized using pentobarbital (6.5 mg/100 mg body weight,
intraperitoneally). An extensive laparotomy was performed under
sterile conditions. The bowel was reflected to one side to expose the
abdominal aorta. The aorta was punctured using a venipuncture catheter. Blood was collected in sterile syringes containing citrate, phosphate, dextrose, and adenine anticoagulant (CPDA-1) and transferred to sterile plastic bags. The blood was then centrifuged at
5,000 × g for 5 min, and the plasma separated. Samples of plasma and
RBCs were cultured to confirm that stored RBCs exhibited no bacterial contamination. The shed blood was stored as packed RBCs (Hct:
70 to 75%) at 4° C. Plasma was frozen and stored at 
40° C.
Preparation of "Fresh" RBCs, "Old" Stored RBCs, and DCLHb
For transfusion of "fresh" RBCs and "fresh" diluted RBCs, packed RBCs stored < 6 d were used, whereas "old" RBCs were defined as packed RBCs stored for 28 to 35 d. For preparation of "fresh" diluted RBCs, packed RBCs were diluted with rat plasma to a hematocrit of 30% (Hb: 100 g/L) 1 to 2 h before transfusion. DCLHb was prepared by Baxter Healthcare Corporation (Round Lake, IL) as described by Chatterjee and colleagues (1). DCLHb was formulated at a concentration of 100 g/L in a lactated electrolyte solution.
Measurements and Calculations
Systemic O2 uptake was measured semi-continuously by means of an Oxymax System (Columbus Instruments, Columbus, OH). A constant flow of room air at a rate of 3.5 L/min was delivered into an airtight box. Gas from the outlet limb was sampled by a paramagnetic O2 sensor for analysis of O2 content and then by an infrared CO2 analyzer. Reference measurements were made by sampling room air every five samples. Systemic O2 uptake was measured from the reduction of air O2 content within the closed system and displayed online. Five consecutive values obtained over a 60-s measurement period were averaged to determine systemic O2 uptake at an individual time point.
Mean arterial pressure and central venous pressure were measured with Uniflow disposable transducers (Baxter Corporation, Toronto, Ontario, Canada) and a Hewlett-Packard 78353B monitor (Hewlett-Packard, Mississauga, Ontario, Canada). Cardiac output was measured by the thermodilution technique using 0.3 ml of normal saline at room temperature injected via the jugular catheter. The thermocouple output was analyzed with a Cardiotherm 500 AC-R cardiac output computer (Columbus Instruments).
Hemoglobin and arterial O2 saturation were assessed using a cooximeter (OSM2b hemoximeter; Radiometer, Copenhagen, Denmark), and lactate concentration was determined by means of a quantitative, enzymatic method (Paramax Analytical System; Baxter Corporation, Mississauga, Ontario, Canada).
Venous O2 saturation, systemic O2 extraction, and systemic O2 delivery were calculated using standard formulas.
Validation of Spectrophotometrically Measured O2 Saturations
The analysis of O2 saturations from a blood substitute/rat blood mixture with a cooximeter is problematic, since the light absorption spectrum of DCLHb is identical to that of unmodified human hemoglobin
but the device can only be calibrated for one blood type (rat blood in
this study). In addition, the OSM2b hemoximeter uses only two wavelengths (506.5 and 600.0 nm) to make measurements. Therefore, the
OSM2b hemoximeter was validated against a second cooximeter (IL
482; Instrumentation Laboratory, Lexington, MA) that measures arterial saturation using four different wavelengths (535.0, 582.2, 594.5, and 626.6 nm) to demonstrate agreement between two independent
measurements. O2 saturations of rat blood and 1:1 DCLHb/rat blood
mixture (thus approximating the mixture of DCLHb and rat blood in
the in vivo experiments) were measured at five different PO2 values.
Since DCLHb contains methemoglobin (MHb; 3 to 4% in fresh solution, increasing at 0.3%/mo), MHb content was also determined. With
the DCLHb/rat blood mixture, curves were rightshifted compared to
rat blood (Figure 1)
this was expected since the DCLHb solution as
evaluated in this experiment was acidotic (pH of 6.9), the pH of the
DCLHb/rat blood mixture was 7.21, and the pH of the whole blood
was 7.4. In comparison to the IL 482 cooximeter, a 3 to 4% rightward
shift was observed with the OSM2b hemoximeter for the range of saturation pertinent to this study (80 to 100%). This may be attributed to
MHb admixture, since the IL 482 cooximeter is designed to "subtract"
MHb admixture (by measuring the partial saturation of oxyhemoglobin), while the OSM2b reads lower saturation values with increasing
MHb content (information from the manufacturer). MHb levels in
three additional in vivo experiments performed using the study protocol for the DCLHb group and fresh DCLHb solution were 0.2, 0.2, and 0.4% before and 0.2, 1.3, and 1.9% after infusion, suggesting that
MHb-related underestimation of the arterial O2 saturation in vivo was
smaller than the 3 to 4% error observed in vitro.
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Postmortem Lung Examination
After euthanization with an overdose of pentobarbital (65 mg), the chests of six animals in the fresh diluted RBC group and five animals in the DCLHb group were opened and the lungs and heart dissected and removed. A double suture was tied around the left hilum, and the left lung was surgically separated. The separated lung was weighed directly and then again after 48 h of exposure to a heat lamp. Wet/dry ratios were calculated as initial weight divided by weight after 48 h. After separation of the left lung, a 14-gauge plastic catheter was advanced into the trachea. The catheter was tied to avoid leakage into the proximal part of the trachea. A total of 5 ml of 0.9% saline was injected into the right lung. After 3 s, fluid was withdrawn and analyzed for hemoglobin content.
Statistics
For statistical analysis, Sigma Stat 1.0 software (Jandel Corporation, San Rafael, CA) was used. Using pilot data, the sample size was calculated to detect a 25% difference in systemic O2 uptake with an alpha of 0.05 and a beta of 0.2. Since randomization was performed after the hemodilution procedure was finished, data from all four groups were combined for the assessment of the effects of hemodilution. Analysis of hemodilution effects was performed using a paired t test. For analysis of survival, the Fisher exact test was applied. In respect to the effects of transfusion, the principal endpoint was the determination of the effects of DCLHb compared to treatment with other blood products. For this reason, an analysis was performed appropriate for a two-factor repeated-measures design with repeat on one factor and comparing DCLHb to the remaining groups. Since the data were unbalanced within groups, a general linearized model was chosen. Wet/ dry ratios were analyzed using the Mann-Whitney rank sum test. For all statistical tests, significance was assumed at a p value < 0.05. Data are presented as mean ± SEM.
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DISCUSSION
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Twenty-four hours after cecal ligation and perforation, all animals demonstrated reduced activity, piloerection, and exudation around the eyes and the nose. Postmortem examination confirmed generalized peritonitis with necrosis of the cecum and significant amounts of purulent secretions in the peritoneal cavity.
Effects of Hemodilution
Hemodilution to O2 supply dependency was accompanied by a fall in hemoglobin (69% from baseline), systemic O2 delivery (72% from baseline), and systemic O2 uptake (46% from baseline; all p < 0.0001) (Table 1). In response to this decrease in tissue O2 delivery, a simultaneous increase in O2 extraction and a decrease in venous O2 saturation (both p < 0.0001) was noted. Both the mean arterial blood pressure (p < 0.0001) and the cardiac index (p < 0.0001) fell by the end of hemodilution, while the lactate increased (p < 0.0001).
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Effects of Treatment during O2 Supply Dependency
With infusion of fresh RBCs, fresh diluted RBCs, and DCLHb, 11 of 12 animals (92%) in each group were resuscitated from O2 supply dependency. In contrast, only four of 12 animals (33%; p < 0.01 versus other groups) survived the study period after the infusion of old RBCs. Since the number of animals remaining in the old RBC group was too small to obtain meaningful results for other transfusion effects, they were excluded from further analysis.
Systemic O2 uptake increased significantly (p < 0.001) in all remaining experimental groups, from 1.62 ± 0.11 to 2.68 ± 0.28 (95% CI: 2.05, 3.31) ml/100g/min with fresh RBCs, from 1.48 ± 0.08 to 2.38 ± 0.16 (95% CI: 2.02, 2.74) ml/100g/min with fresh diluted RBCs, and from 1.43 ± 0.07 to 1.96 ± 0.17 (95% CI: 1.58, 2.34) ml/100g/min with DCLHb (Figure 2). The positive effect of all three groups on systemic O2 uptake was accompanied by an increase in systemic O2 delivery following both fresh RBC and fresh diluted RBC infusion. In contrast, systemic O2 delivery did not increase following the DCLHb infusion (p < 0.0001 compared to other groups; Table 2, Figure 3). This may, for the most part, be explained by only a small increase in hemoglobin concentration after DCLHb infusion (DCLHb: 39.4 ± 3.7 to 50.3 ± 2.6 g/L; fresh diluted RBCs: 37.8 ± 2.4 to 63.6 ± 2.3 g/L; fresh RBCs: 40.1 ± 2.3 to 129 ± 2.5 g/L; p < 0.0001 DCLHb compared to other groups). There was also a significant decrease in arterial O2 saturation following the infusion of DCLHb, while the arterial O2 saturation was well preserved in the other study groups (p < 0.0001; see Table 2).
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significance for treatment
effect between groups DCLHb versus fresh diluted RBCs; 
significance for treatment effect between groups DCLHb versus fresh
RBCs (ANOVA).
Further effects of the infusion of the fresh RBC preparations and DCLHb are summarized in Table 2. Following both fresh RBC and fresh diluted RBC infusion, systemic O2 extraction fell, while the infusion of DCLHb was accompanied by a further increase in systemic O2 extraction (p < 0.0001 compared to the other groups). An additional paired comparison between O2 extraction before and after DCLHb infusion revealed a significant increase in O2 extraction within the DCLHb group (p < 0.05). Calculated venous O2 saturation increased after infusion of the fresh blood preparations, while it further decreased after DCLHb (p < 0.0001 DCLHb versus the other groups). Mean arterial pressure recovered with all three treatments, although this effect was less distinct with DCLHb than with fresh RBCs (p < 0.01). Arterial lactate concentrations also decreased significantly with all treatments. Cardiac index rose following fresh diluted RBC infusion, but it remained unchanged with DCLHb administration (p < 0.05 between groups).
Because of the depressed arterial O2 saturations during the DCLHb infusion, we undertook additional studies to examine for potential pulmonary capillary leakage of DCLHb. In bronchoalveolar lavage of animals receiving fresh diluted RBCs (n = 5) and DCLHb (n = 6), no hemoglobin was detected. The medians of lung wet/dry ratios obtained after postmortem extirpation were comparable (5.1 for fresh diluted RBCs versus 5.2 for DCLHb).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Although numerous studies have characterized the effects of DCLHb on cardiovascular performance and organ perfusion, the effects of DCLHb on O2 delivery and uptake have not been explicitly investigated. Therefore, we determined the ability of DCLHb to improve systemic O2 uptake when the tissues were in a state of O2 need. In rats made septic by cecal ligation and perforation, isovolemic exchange infusion of DCLHb during O2 supply dependency was accompanied by an acute increase in systemic O2 uptake.
Background
Since O2 supply-demand imbalance contributes to the tissue injury underlying multiple-organ dysfunction in sepsis, it is not surprising that blood substitutes with O2-carrying properties are being proposed as therapeutic agents in this syndrome (17). In rats made septic by peritoneal injection of cecal slurry, DCLHb prevented leukocytosis, induced transient hypertension, and also reduced mortality from sepsis (17). This study also showed that infusing DCLHb after development of sepsis-related cardiovascular dysfunction improved blood flow to organs such as heart, brain, stomach, cecum, colon, and spleen.
To demonstrate the efficacy of RBCs or a RBC substitute, the choice of an appropriate and relevant endpoint is crucial. When addressing efficacy in relation to RBC transfusion, surrogate endpoints such as the demonstration in preserved cells of acceptable levels of 2,3-DPG and p50 and > 80% in vivo survival after transfusion (11, 12) are most commonly used. Recently, post-transfusion O2 content and hematocrit have also been proposed as measures of efficacy (22). However, if the property to be examined is O2 carriage, such surrogates are not likely appropriate as efficacy endpoints for RBC substitutes (22). In clinical settings, on the other hand, reliable evidence for the efficacy of O2 carriers without the use of surrogate endpoints is difficult to obtain. For example, attempts to demonstrate the efficacy of RBCs in septic patients by measurement of systemic O2 uptake using indirect calorimetry have not been successful (23).
Therefore, we have developed an animal model with an unequivocal "signal" to measure efficacy of treatments directed at modifying tissue oxygenation (20). A very important feature of this model is that it provides direct measurements of systemic O2 uptake. Using isovolemic hemodilution to depress systemic O2 delivery in awake rats, we establish supply dependency of systemic O2 uptake, a point where the tissues are in a state of acute O2 need. As previously demonstrated (20), the efficacy of treatments intended to increase O2 availability, such as transfused RBCs and catecholamine infusions, is concluded if systemic O2 uptake increases following treatment. We avoid anesthesia in this approach since such agents may independently modify the circulatory compensation to acute changes in O2 delivery (24).
Comparing DCLHb, Fresh RBCs, and Aged RBCs
We compared the effects of DCLHb to packed RBCs that had been hemodiluted to the same hemoglobin content as that of infused DCLHb. Freshly stored packed RBCs were assumed to be the most efficient RBC preparation that is currently available and, therefore, comprised a third treatment group for comparison. Since aged RBCs were reported to cause adverse effects in sepsis (23), another treatment group for comparison to DCLHb was RBCs that had been stored in CPDA-1 for 28 to 35 d. A second rationale for inclusion of a group of animals receiving aged RBCs was to balance the comparisons with regard to the clinical situation, where patients requiring RBC transfusion receive RBC units stored for different times rather than "fresh" cells. As previous experiments demonstrated that placebo infusion alone was followed by early death in four of five experiments, we discarded the addition of another comparative group with a placebo infusion (i.e., normal saline) for ethical reasons. RBCs were prepared and stored according to standard blood bank conditions. Previous studies from our laboratory determined that storage of rat blood in CPDA-1 for up to 28 d was accompanied by both 2,3-DPG depletion and 24-h survival of 80% of RBCs after transfusion (25).
As expected, exchange transfusion with fresh, hemodiluted RBCs acutely elevated the calculated systemic O2 delivery, while O2 extraction fell. Exchange transfusion with DCLHb also acutely elevated the systemic O2 uptake, restored the mean arterial pressure, and lowered the arterial lactate concentration, which had been elevated under O2 supply-dependent conditions. This observation is interpreted as confirmation of the ability of DCLHb to improve tissue O2 availability in our model of sepsis. However, a limitation of this study is that the sample size was too small to exclude a significant difference for systemic O2 uptake when comparing the DLCHb group to the other two groups, as shown by the confidence intervals post-transfusion. This may be especially important for the comparison against the fresh blood group, since here the differences were substantial enough to be of physiologic relevance.
Somewhat surprisingly, the improved tissue O2 uptake following DCLHb occurred without an increase in calculated systemic O2 delivery. The effects of the RBC preparations and DCLHb on the systemic O2 uptake/delivery relationship in this study are shown in Figure 4. To our knowledge, this study is the first study to demonstrate support of systemic O2 uptake by increasing O2 extraction in a supply-dependent state. This may suggest that the O2 uptake/delivery relationship can be improved even under supply-dependent conditions. This finding is supported by data of Standl and associates (26), who reported that in skeletal muscle of hemodiluted dogs augmentation of 0.7 g/dL bovine hemoglobin restored tissue O2 tension in hypoxic areas to a much higher extent than the same dose of either fresh or aged blood.
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Changes in calculated systemic O2 delivery represent direct proportional changes in cardiac index, arterial O2 saturation or hemoglobin content. Although arterial O2 saturation fell modestly following DCLHb infusion and the cardiac index was not augmented, it appears that the lack of increase in O2 delivery following DCLHb as compared to fresh RBC was mainly due to the only small increase in arterial hemoglobin. The lack of a clear increase in arterial hemoglobin concentration may be attributed to the short intravascular half-life of DCLHb, which is 4 h in septic rats (27), the use of exchange transfusion, and colloidal properties of this agent resulting in further hemodilution.
What are the possible explanations for an improved tissue O2 utilization following DCLHb infusion without a parallel increase in convective systemic O2 delivery? First, DCLHb could have mediated a redistribution of blood flows from organs not fully supply dependent towards organs that were supply dependent. It is also possible that DCLHb altered the intraorgan distribution of blood flow by reversing sepsis-induced abnormalities in microvascular perfusion. Both improved inter- and intra-organ distribution of blood flow would be followed by a better match between tissue O2 needs and O2 supply, thereby explaining the increase in O2 extraction following DCLHb infusion.
Further, improved O2 extraction with DCLHb may be related to a more homogeneous intravascular distribution of
cell-free hemoglobin as compared to RBCs. Even in health,
there is considerable heterogeneity in RBC density among individual capillaries, with some capillaries receiving high RBC
supply, while others have lower RBC supply with longer intervals of pure plasma flow (28). After hemodilution, as in the
current study, RBC supply to the individual capillaries will be
further reduced. Therefore, extreme hemodilution might be
a situation in which cell-free hemoglobins are highly efficaciousespecially in capillaries with low RBC supplybecause
of hemoglobin presence in RBC-free plasma gaps. Evidence
for such a mechanism was recently demonstrated using a bovine hemoglobin (26).
Another possibility is that a right shift of the O2 dissociation curve (see Figure 1) may have facilitated O2 release to the tissues and thus augmented O2 extraction in DCLHb-treated animals. This right shift could be due to decreases in pH and/ or differences in p50 between rat hemoglobin and DCLHb, which is derived from human hemoglobin.
A right-shifted O2 dissociation curve may also contribute to the decrease in arterial O2 saturation after DCLHb infusion and could thus, in addition to the increase in O2 extraction, have mediated the fall in venous O2 saturation in animals that received DCLHb. Lower arterial O2 saturation also may have been due to impaired gas exchange resulting from pulmonary edema or ventilation perfusion mismatch. In this study, there was no evidence of pulmonary edema based on the wet/dry ratio and bronchoalveolar lavage data. Binding of NO by DCLHb could potentially aggravate ventilation perfusion mismatch in this model, thus elevating the intrapulmonary shunt. This possibility is supported by reports that crosslinked hemoglobin, due to its NO-scavenging properties, induced pulmonary hypertension, and attenuated gas exchange in an animal model of sepsis (29). An alternative explanation is suggested by recent data showing that DCLHb perfuses narrowed lung capillaries that are inaccessible to RBCs (30). Thus, DCLHb may have increased perfusion of areas of the lung that were underventilated because of pathologic changes associated with this model.
This study also demonstrates an increased mortality following transfusion of old stored RBCs in septic rats placed in O2 supply dependency. This finding is in principal agreement to previous studies that reported a failure of aged RBCs to increase tissue O2 uptake in the same rodent model (20). However, our data do not provide direct information about O2 uptake by tissues from aged red cells.
Adverse effects of aged RBCs have also been reported in a clinical trial. In a series of 23 critically ill septic patients who received three units of packed RBCs, splanchnic ischemia occurred in those patients receiving RBCs stored for more than 15 d (23).
The deleterious effects of aged RBCs in sepsis may be due
to the physical and biochemical changes in stored blood, collectively termed the "storage lesion," and to interactions with
sepsis-induced cytokine responses and the injured microcirculation. The storage lesion includes decreased 2,3-DPG and
ATP levels, acidosis, swelling, and shape and deformability
changes of the red cell (31). Shape and deformability changes
have been shown to promote red cell trapping in the microcirculation, hence impeding blood flow and increasing systemic
vascular resistance even in healthy subjects (32, 33). In sepsis,
endotoxin and cytokines such as tumor necrosis factor-
, interleukin-1
, and antioxidants were reported to stimulate adhesion of RBCs to the endothelium (34). The immunologic response to sepsis might, in addition to microcirculatory injury such as impaired microvascular RBC flux (35), have further
potentiated adverse effects of poorly deformable aged erythrocytes in the current study.
Summary
The increase observed in O2 uptake and the reversal of lactic acidosis during O2 supply dependency in septic rats clearly demonstrated a beneficial effect of DCLHb on O2 utilization by the tissues. This benefit of DCLHb was most likely the consequence of an improved O2 extraction. For the treatment of sepsis, support of tissue oxygenation and treatment of circulatory failure are two major objectives. Since DCLHb might be useful for both, it offers new therapeutic possibilities in this syndrome.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Dr. W. J. Sibbald, London Health Sciences Centre, Victoria Campus, 375 South St., London, ON, N6A 4G5 Canada.
(Received in original form September 19, 1996 and in revised form May 5, 1997).
Acknowledgments: The writers wish to thank Dr. M. d'Almeida for preparing the RBC solutions and facilitating appropriate randomization. Many thanks also to M. White for her excellent technical assistance in the experimentation.
Supported by grants from the Baxter Healthcare Corporation (Round Lake, IL) and the government of Ontario, Canada (University Research Incentive Fund).
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References |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES
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1. Chatterjee, R., L. V. Welty, R. Y. Walder, S. L. Pruitt, P. H. Rogers, A. Arnone, and J. A. Walder. 1986. Isolation and characterization of a new hemoglobin derivative cross-linked between the a chains (lysine 99a1, lysine 99a2). J. Biol. Chem. 21: 9929-9937 .
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Vandergriff, K. D.,
F. Medina,
M. A. Marini, and
R. M. Winslow.
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264:
17824-17833
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