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1Cardiovascular Pulmonary Research Group, School of Medicine; 2Department of Anesthesiology, University of Colorado Health Science Center, Aurora, Colorado; 3Department of Health and Exercise Science; and 4Department of Biomedical Sciences School of Veterinary Medicine, James L. Voss Veterinary Teaching Hospital, Colorado State University, Fort Collins, Colorado
Submitted 20 March 2008 ; accepted in final form 17 June 2008
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ABSTRACT |
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hemoglobin-based oxygen carriers; Nitric oxide; Cardiac output
HBOCs that are of clinical interest currently constitute several types, based on the chemical modification of the hemoglobin molecules to provide longer circulation times and optimal oxygen affinity and decrease negative side effects. These HBOC types have been reviewed recently by Winslow (26) and are categorized into cross-linked, polymerized, and conjugated hemoglobin. Cell-free hemoglobin has long been associated with hypertension when infused (26). Almost all polymerized hemoglobin variants induced variable degrees of vasoconstriction (9, 15, 20). Although the cause is still debated, most HBOC vasoconstrictive properties likely stem from nitric oxide scavenging (17, 23). In contrast, Tsai et al. (23) demonstrated that polyethylene-glycol-decorated polymerized Hb had similar nitric oxide affinity as other polymerized Hb derivatives but did not produce vasoconstriction. Thus, this supports the alternative theories that vasoconstriction may be a result of supraphysiological oxygenation of tissues surrounding blood vessels, dependent on the molecular volume of the oxygen binding sites on HBOCs (26), or due to an oncotic volume expansion effect (14).
HBOC therapies have been primarily studied for blood loss treatment (7, 12, 15, 24), and studies investigating HBOC as a means for enhanced oxygen therapy, other than for blood loss treatment, are few (16, 18). Unfortunately, these studies either focused on the hemodynamics or on the oxygen binding characteristics of HBOC but failed to comprehensively provide data on the influence of HBOC-induced hemodynamic changes on oxygen delivery per se. HBOC infusion in euvolemic individuals would likely occur during radiation and chemotherapy therapy, pedicled flap treatment, and cerebral ischemia to enhance tissue oxygen delivery in such conditions.
We hypothesized that administration of a gluteraldehyde-polymerized bovine hemoglobin (PBvHb) to euvolemic animals during acute global hypoxia would increase the oxygen carrying capacity of blood and oxygen delivery to tissues, despite the possibility of PBvHb-induced vasoconstriction and other hemodynamic changes, such as reduced cardiac output. PBvHb was chosen for this study, as this HBOC has been approved for veterinary use in the United States and South Africa and for limited use in humans in South Africa.
Our primary goal was to determine the oxygen delivery and systemic and pulmonary hemodynamic effects after a 3-ml infusion of PBvHb during acute hypoxia compared with normoxia in a conscious rat model of global hypoxia. Therefore, studies were designed to measure oxygen content, blood gases, and pulmonary and systemic hemodynamics in awake, conscious rats exposed to either 21 or 10% oxygen. Secondarily, the study was designed to determine the potential role of nitric oxide in PBvHb-induced vasoactivity.
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METHODS |
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Male Sprague-Dawley rats (280–350 g and 10–12 wk of age) were obtained from a commercial vendor (Charles River) and housed in the University of Colorado Health Sciences Center's Center for Laboratory Animal Care (elevationn = 5,280 ft). Animals were allowed ad libitum access to food and water and were kept on a 12-h day-night cycle. All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at University of Colorado Health Sciences Center.
Instrumentation
Rats were allowed to acclimate to Denver altitude (5,280 ft; 1,690 m; PB 
630 mmHg) for at least 7 days before instrumentation. Forty-eight hours before surgery, rats were provided water supplemented with acetaminophen/codeine (0.5 mg/ml and 0.05 mg/ml, respectively) for postoperative analgesia. The animals were weighed, and the hematocrits were determined. Rats were anesthetized with a mixture of ketamine: Rompon (xylazine; 75 mg/kg: 6 mg/kg ip). Under aseptic conditions, the left carotid artery was cannulated with a PE-50 (0.58 mm ID, Becton Dickinson) catheter. A PV-1 (0.28 mm ID, Becton Dickinson) catheter with a shallow bend at its tip was inserted into the right ventricle via the right jugular vein and guided into the main pulmonary artery. Pressure tracings confirmed placement in the pulmonary artery. Next, two PE-50 (0.58 mm ID, Becton Dickinson) catheters were placed in the superior vena cava via the right jugular vein for venous blood collection and to obtain cardiac output values. All catheters were flushed with heparinized saline, tied off, tunneled subcutaneously to the dorsal neck region, and exteriorized at the back of the neck. Animals were allowed at least 48 h to recover before any treatments. Animals demonstrating signs of infection, diarrhea, or distress were excluded from study.
Estimation of Blood Clearance Rate and Physical Characteristics of PBvHb
It has previously been suggested that the PBvHb plasma concentration half-life is species and dose dependent (16, 13). Therefore, the PBvHb clearance rate was estimated in male Sprague-Dawley rats (n = 4) infused with 1.3 g/kg PBvHb. Arterial blood was collected hourly for 8 h postinfusion, and plasma PBvHb concentrations were plotted against time to estimate the PBvHb clearance rate in the rat. It was determined that the concentration was reduced by approximately one-half at 5 ± 1 h and undetectable in the blood stream by co-oximetry 24 h after infusion (data not shown). Therefore, in the present study, the PBvHb effects were measured at 2 and 4 h, followed by effects of nitric oxideS inhibition at 4.5 h postinfusion. The physical characteristics of PBvHb "Oxyglobin" are shown in Table 1.
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Animals were randomly assigned into one of four groups, each with a sample size of 10 animals (n = 10) unless otherwise specified: 1) normoxic (FIO2 = 21% O2) lactated Ringer solution (Henery Schein, Melville, NY) infused (Normoxia-R); 2) normoxic polymerized bovine hemoglobin (Biopure, Cambridge, MA, approved for veterinary use; Table 1) infused (Normoxia-PBvHb); 3) hypoxic (FIO2 = 10%) lactated Ringer solution infused (Hypoxia-R); and 4) hypoxic polymerized bovine hemoglobin infused (Hypoxia-PBvHb).
Hemodynamic Measurements
Rats were placed in a custom designed small, rectangular, plexiglas chambers with a portal through which catheters could be passed. Catheters were flushed with heparinized saline and then connected to fluid filled pressure transducers. Animals were exposed to normoxia and hypoxia by flushing the chamber with appropriate gases. Once breathing hypoxic gas, hypoxic animals were not reexposed to room air. Then, blood pressures, pulmonary artery blood pressures, heart rates, and cardiac outputs of normoxic and hypoxic animals were collected before Ringer or PBvHb treatment (Fig. 1).
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Blood Gas and Co-Oximetry Measurements for Whole Blood and Plasma
Blood samples were collected immediately after hemodynamic measurements had been obtained at each time point (Fig. 1). Arterial (0.2 ml) and venous (0.2 ml) blood was withdrawn via carotid and venous catheters, respectively, into blood gas syringes and analyzed (ABL5, Radiometer, Copenhagen and co-oximetry via OSM3, Radiometer, Copenhagen) with algorithms specific to rat and bovine hemoglobin.
Plasma phase co-oximetry. Heparinized microhematocrit capillary tubes (100 µl; Fisherbrand, #22362566) were immediately filled from each 1-ml blood gas syringe (see above), sealed from room air with a removable cap (Kendall Healthcare, #8889212000), and spun on a capillary centrifuge to separate cell and plasma fractions. The plasma portion of the capillary tube was aspirated into the co-oximeter. Measurements were excluded if any portion of the cell fraction was inadvertently aspirated into the analyzer. Care was taken to avoid exposing blood samples to room air.
Calculation for Oxygen Delivery, Consumption, and Extraction
Oxygen delivery (DO2), consumption (
O2), and extraction ratio (ERO2) were calculated for hemoglobin in whole blood and PBvHb in the plasma phase (no measurable endogenous hemoglobin was present in the plasma) with the following mathematical calculations: 1) DO2 = (CaO2) x CI, where CaO2 and CvO2 = (HbO2%/100) x tHb x 
* and HbO2 = HbO2SAT(100 – HbCO – MetHb) fraction*; 2) O2 = [(CaO2) – (CvO2)] x CI; and 3) ERO2 = O2/DO2, where CI is cardiac index and CaO2 and CvO2 are arterial and venous content respectively. is oxygen capacity for rat or bovine hemoglobin. Asterisks indicate OSM3 programmed algorithms.
Statistics
For all groups, means ± SE are reported. Statistical comparison between groups were analyzed with either a multifactorial (FIO2, PBvHb-treatment) with repeated measures (Time) ANOVA (see Tables 1, 3–4) or a multifactorial (FIO2, PBvHb-treatment, NLA-treatment) ANOVA (see Figs. 3–5; Table 2). Unless otherwise noted, the 2- and 4-h time points were combined for determination of either a PBVHB or NLA treatment effect for all hemodynamic values. Post hoc analyses were completed with unpaired, two-sided Student's t-test with a Bonferroni adjustment. Statistical analyses were performed using JMP (Version 5) statistical software package (SAS; Cary, North Carolina) with statistical significance set at P 
0.05.
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RESULTS |
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Normoxia.
As expected, the total Hb concentration in whole blood was lower after a 3-ml infusion of Ringer solution due to dilution (P < 0.01; Table 2). On the other hand, addition of 3 ml of PBvHb increased the total Hb concentration 10% (P < 0.02; Table 1), due to the presence of PBvHb in the plasma (2 g/dl; Table 2). An elevation of methemoglobin concentration accompanied the rise in total Hb concentration in whole blood and plasma (P < 0.04; Table 1). Remarkably, the methemoglobin concentration in plasma increased approximately sixfold between 2 and 4 h in PBvHb-treated animals (P = 0.03, 2 vs. 4 h; Table 2).
Hypoxia. Hypoxia (4 h) per se had no effect on either total or plasma Hb concentration. However, total methemoglobin concentration (P < 0.001; Table 1) was lower during hypoxia compared with normoxia. This observation held true for the plasma methemoglobin concentration (P = 0.04) of PBvHb-treated animals as well (Table 2). Although plasma methemoglobin concentration increased in PBvHb-treated animals between 2 and 4 h during normoxia, no change between these time points was noted during hypoxia. (Table 2).
Blood Gases
Arterial PO2, PCO2, and pH. Neither Ringer nor PBvHb altered PO2, PCO2, or pH during normoxia or hypoxia. However, hypoxia produced expected decreases in PO2 and PCO2 and increases in pH (P < 0.001; Table 2).
Oxygen Content and Saturation
Normoxia.
Whole blood oxygen content was lower after dilution by Ringer infusion (P = 0.01; Table 2). Treatment with PBvHb did not improve whole blood oxygen content, yet oxygen content was measurable in the plasma phase (1.5 mmol; Table 2). Whole blood oxygen saturation was unaffected by either Ringer or PBvHb treatment. In PBvHb-treated animals, oxygen saturation of the plasma phase was lower than oxygen saturation in the whole blood (P < 0.001; Table 2).
Hypoxia. As expected, hypoxia decreased oxygen content and saturation in whole blood and plasma (P < 0.001; Table 2) before treatment. PBvHb-treated animals had significantly greater whole blood oxygen content within 2 h of hypoxia (P = 0.005; Table 2).
Hemodynamics
Unless otherwise stated Ringer infusion had no effect on hemodynamic variables in animals exposed to either normoxia or hypoxia.
Mean arterial pressure and total peripheral resistance.
NORMOXIA.
The mean arterial pressure (MAP) and total peripheral resistance (TPR) were elevated from untreated values by PBvHb treatment (P < 0.001; Fig. 2, A and B; Table 3). The NLA-induced increase in pressure and resistance in Ringer-infused animals was equivalent to the elevation in MAP and TPR caused by PBvHb alone (P < 0.001; Fig. 2, A and B; Table 4).
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Mean pulmonary artery pressure and total pulmonary resistance.
NORMOXIA.
We observed no change from untreated values in mean pulmonary artery pressures in animals treated with PBvHb, but a significant increase in total pulmonary vascular resistance was noted (P < 0.001; Fig. 3, A and B; Table 3). Treatment with NLA had no effect on mean pulmonary artery pressures in either the Ringer or PBvHb groups (Fig. 3, A and B; Table 4). However, NLA increased pulmonary vascular resistance in the Ringer-infused group to a similar extent as that observed after PBvHb treatment (P = 0.04; Fig. 3B; Table 4).
HYPOXIA.
As expected, mean pulmonary artery pressure was increased with hypoxia exposure (Table 3). The initial increase in pulmonary artery pressure induced solely by hypoxia was further increased with PBvHb treatment, along with an increase in pulmonary vascular resistance (P < 0.001; Fig. 3, A and B; Table 3). The pulmonary artery pressure and pulmonary vascular resistance were increased further by NLA in the Ringer-infused animals (P = 0.03; Fig. 3, A and B; Table 4). However, NLA had no additional influence on the PBvHb-induced increase in pulmonary hemodynamics (Fig. 3, A and B).
Heart rate and pulse pressures.
NORMOXIA.
Infusion of Ringer or PBvHb solution caused a brief but transient decrease in heart rate that was resolved by 4 h (P = 0.02 for Ringer and P = 0.003 for PBvHb; Fig. 4A; Table 3). However, pulse pressure decreased from untreated values after PBvHb but not with Ringer treatment (P < 0.001; Table 1). NLA decreased HR and pulse pressure only in the Ringer-infused animals (P = 0.04 for HR; Fig. 4A; and P < 0.001 for pulse pressure; Table 4).
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Cardiac index and stroke volume. normoxia.
Treatment with PBvHb induced a dramatic decrease in cardiac index and stroke volume (P 0.002; Fig. 4, B and C; Table 3). NLA decreased cardiac index and stroke volume in Ringer-infused animals (P 0.02; Fig. 4, B and C; Table 4), but did not change cardiac index and stroke volume in the PBvHb-treated group.
HYPOXIA.
Hypoxic stress increased the cardiac index (Table 3). However, hypoxia PBvHb treatment greatly decreased cardiac index and stroke volume, as had occurred in normoxia (P = 0.006; Fig. 4, B and C; Table 3). In Ringer-infused animals, treatment with NLA induced a decrease in cardiac index and stroke volume similar in magnitude to that observed in PBvHb-treated animals (P 0.02; Fig. 4, B and C; Table 4). Interestingly, NLA further decreased cardiac index and stroke volume in the PBvHb animals (P < 0.001; Fig. 4, B and C; Table 4).
Oxygen delivery, Consumption, and Extraction
Normoxia.
Treatment with PBvHb reduced oxygen delivery and O2 (P < 0.001; Fig. 5; Table 5), but Ringer infusion did not. Thus the calculated extraction rate for oxygen was less with PBvHb compared with Ringer treatment during normoxia. The reduction in oxygen extraction from whole blood was greater than that from plasma (P < 0.001; Table 5).
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O2 in all animals (P < 0.001; Table 5). However, in Ringer-infused animals O2 had returned to normoxic values by 4 h, while PBvHb treatment during hypoxia lowered O2 even below that in normoxic PBvHb animals.
In all animals, hypoxia induced a rise (18%) in whole blood oxygen extraction ratio over the time course of the study period (P = 0.038, untreated vs. 2 and 4 h pooled; Table 4). However, in PBvHb-treated animals, the oxygen extraction ratio for plasma was lower during acute hypoxia than during normoxia but doubled between the 2- and 4-h time periods (P = 0.02; Table 4).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The HBOC used in this study consisted of purified gluteraldehyde cross-linked PBvHb. The species differences, purity, and/or specific cross-link may have contributed to some of the adverse effects seen in this rat model. For example, compared with other products, HBOCs prepared from human hemoglobin cross-linked with pyridoxal-5'-phosphate followed with polyoxyethylene glycol have been shown to be less vasoactive in exchange transfusion models (1, 2).
The time course of the experiments and the infused concentration of PBvHb were selected based on estimated HBOC clearance rate (see METHODS) and the in situ concentration of PBvHb necessary to increase the hypoxic blood oxygen content to that of the normoxic oxygen content levels. Clearance rates in the current study are similar to those previously published for mice (16). The volume (3 ml) of PBvHb or Ringer solution infused over a 30-min period did not cause hemodynamic changes and could be safely infused without adverse effects due to volume overload, as has been previously reported (21). The PBvHb dose (1.3 g/kg) was one- to threefold higher than what others have used in hypervolemic models (6, 23). However, the goal of these previous studies (6, 23) was to study the reaction between various HBOC and nitric oxide and not to increase oxygen content to compensate for hypoxia. Interestingly, Tsai et al. (23) suggested that the microvascular effects of HBOC may not be based on concentration but on HBOC properties such as molecular modification, molecular volume, and oxygen affinity. In fact, data indicate that as little as 0.5 g/dl of HBOC is sufficient to scavenge most intravascular nitric oxide (23). Therefore, the larger dose of PBvHb used in the current study to compensate for a hypoxia-induced reduction in arterial oxygen content should not have had an additional nitric oxide scavenging effect.
Blood Gas Variables
A primary goal of this study was to determine whether or not PBvHb infusion could enhance blood oxygen content sufficiently enough to compensate for low arterial oxygen saturation and content during acute hypoxia. As expected PBvHb infusion increased total hemoglobin and arterial oxygen concentration in whole blood by 10% in all animals. The difference in whole blood and plasma hemoglobin oxygen saturation from the current study is in agreement with previous findings in which cross-linked PBvHb have shown altered oxygen binding characteristics (3). However, our data showed that increased total hemoglobin and oxygen content tracked together at 10% after PBvHb infusion. This would suggest that the HBOC oxygen binding was sufficient to maintain an equal relationship between total hemoglobin and oxygen content. Thus although PBvHb has different oxygen binding characteristics than native hemoglobin, PBvHb infusion did increase oxygen content during hypoxia sufficiently to offset the loss of oxygen content that occurred during hypoxia exposure.
HBOC Hemodynamic Effects During Normoxia
Because oxygen delivery is determined by oxygen content and cardiac output, the finding that oxygen content was improved with PBvHb but cardiac output was markedly decreased in normoxia is of importance to therapeutic considerations. This is the first study to report such a large fall in cardiac output with PBvHb infusion during normoxia. The finding that PBvHb infusion increased MAP and TPR in normoxic conditions is in agreement with other studies (6, 14, 17, 23) demonstrating an PBvHb-induced systemic pressor response. It is likely that greater MAP and peripheral resistance increased ventricular afterload, decreased heart rate and stroke volume, and thus impaired cardiac output (8).
Because inhibition of nitric oxide synthase in normoxia did not cause a further rise in systemic resistance or cardiac output, it is likely that nitric oxide scavenging by PBvHb was ultimately responsible for the reduction in cardiac output. It is also possible that PBvHb had a direct effect on cardiac function by inducing cardiac lesions, though this has not been demonstrated in the rat, compared with other species (4). However, investigators (4) did not demonstrate that the heart lesions directly decreased cardiac index. Overall, it appears that during normoxia, nitric oxide scavenging by PBvHb increased left ventricular afterload, lowering cardiac index.
PBvHb Hemodynamic Effects During Acute Hypoxia (FIO2 = 10%)
During hypoxia, PBvHb caused a further reduction in oxygen delivery despite the maintenance of oxygen content. This was directly related to a PBvHb-induced decrease in cardiac output similar to that observed in normoxia. However, our data suggest that in hypoxic conditions PBvHb further elevated mean pulmonary artery pressure and subsequently increased right ventricular afterload, likely contributing to the lowering of cardiac output. This reduction in cardiac output occurred in combination with the elevated MAP and left ventricular afterload.
Nitric oxide synthase inhibition further elevated pulmonary artery pressure during hypoxia in Ringer-infused animals but not PBvHb-treated animals. Thus the PBvHb-induced decrease of cardiac output is likely indirectly mediated by vascular nitric oxide scavenging by HBOC during both normoxia and hypoxia; however, the lower cardiac output appears to be primarily due to elevated right ventricular afterload during hypoxia compared with increased left ventricular afterload during normoxia.
An interesting observation regarding hypoxia and systemic pressures requires addressing. Although hypoxia generally increases systemic pressures, the reduction in peripheral vascular resistance and blood pressures in all animals during hypoxia in the present study may be explained by earlier reports in which acute hypoxic insult resulting in arterial PO2 < 40 mmHg (as in the current study) caused locally mediated vasodilation and, thus, decreased arterial blood pressure (11, 25). Although PBvHb treatment (and nitric oxide scavenging) during hypoxia elevated vascular resistance and blood pressure, pressures were not returned to normoxic values after PBvHb treatment. This suggests that the mechanism of hypoxia-induced systemic vasodilation in the present study was not completely nitric oxide mediated. Interestingly, treatment with a nitric oxide synthase inhibitor during hypoxia further elevated blood pressure and vascular resistance in PBvHb but not Ringer-treated animals. There is no clear cause of this phenomenon, and more research is necessary to elucidate this issue.
Mechanisms of PBvHb-Induced Vasoconstriction
Nitric oxide scavenging by HBOC is a primary contributor to rising systemic pressure during normoxia (1, 2, 6, 14, 17, 23), and our data confirm that it also contributes significantly to PBvHb-induced elevation of pulmonary artery pressure during hypoxia in conscious animals. However, PBvHb-induced vasoconstriction could also be influenced by volume expansion due to elevated intravascular oncotic pressure (12) and/or an "autoregulatory" vasoconstrictive effect in which a high P50 Hb favoring rapid oxygen unloading exposing arteriolar vessel walls to supranormal PO2, triggering vasoconstriction (26).
Oxygen Delivery, Consumption, and Extraction
PBvHb impaired oxygen delivery during both normoxia and hypoxia. The 50% reduction in cardiac index with PBvHb infusion accounts for the decreased oxygen delivery as discussed above. Interestingly, during both normoxia and hypoxia, PBvHb decreased oxygen consumption significantly. This is notable, because unless aerobic metabolism is decreased, one would expect oxygen consumption to be maintained near baseline values throughout the 4-h study period. Interestingly, Cabrales (5) observed a similar response in the microvasculature in a Hamster window chamber experiment 1 h after an 80% exchange transfusion. Similar to those experiments, it is hard for us to draw any conclusion as to whether or not this is a positive or negative consequence of PBvHb. Perhaps PBvHb treatment and its vascular effects resulted in the preferential diversion of blood flow from higher to lower oxygen consuming tissue, but this will require further investigation.
Oxygen extraction, a function of oxygen delivery vs. oxygen consumption, was greater in whole blood than in plasma during PBvHb treatment under normoxic and hypoxic conditions. This is in agreement with previous studies of oxygen transport during PBvHb treatment (24). During hypoxia in Ringer-infused animals, as expected, the extraction of oxygen by the tissues (as reflected by the extraction ratios) increased over the 4-h study period to compensate for decreased oxygen delivery, maintaining oxygen consumption. In contrast, with PBvHb treatment during normoxia and hypoxia, the extraction of oxygen by the tissue did not increase sufficiently enough to maintain oxygen consumption. This important observation requires further investigation to determine how PBvHb may reduce oxygen consumption and extraction.
In conclusion, hemodynamic and blood gas data indicate that PBvHb infusion was ineffective as a means for increasing oxygen delivery during acute hypoxic insult. In fact, PBvHb had a negative consequence on oxygen delivery during both normoxia and hypoxia. The primary mechanism for the negative effects of PBvHb during isovolemic hypoxia appears to be nitric oxide scavenging with confounding effects on cardiac output, oxygen extraction, and oxygen consumption. It remains to be determined if eliminating the nitric oxide scavenging properties of PBvHb will enable PBvHb to become an effective oxygen carrier and oxygen deliverer under normoxic and/or hypoxic conditions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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