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Department of Bioengineering, University of California, San Diego, La Jolla, California 92093-0412
Submitted 2 July 2004 ; accepted in final form 3 August 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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blood substitutes; methemoglobin; functional capillary density; microcirculation
Polymerized bovine Hb [Oxyglobin (PBH)] is a highly purified bovine Hb that is inter- and intramolecularly cross-linked with glutaraldehyde and is commercially available as an O2 therapy for use in anemic dogs. PBH consists of a heterogeneous mixture of 32- to 500-kDa polymeric (
95%) and nonpolymeric (5%) species (35). Polymerization with glutaraldehyde is known to alter the O2 affinity, redox potential, and autoxidation kinetics of human Hb (9, 17). Alayash (1) also showed that 50% polymerization with glutaraldehyde can alter the O2-carrying capacity and some of the redox properties of bovine Hb.
The rapid oxidation (autoxidation) of Hb to a nonfunctional ferric [methemoglobin (MetHb)] form is a concern in the use of Hb as an O2 carrier. This uncontrolled and spontaneous oxidation of ferrous iron compromises the function and, in some instances, the safety of the infused Hb blood substitute. In vivo, Hb is localized within red blood cells (RBCs) and does not come in direct contact with tissues and cells. The presence of HBOCs in the circulation, however, raises the possibility of their direct interaction with the endothelium, as well as blood-borne cells, including platelets. Enhancement of platelet deposition and accelerated thrombosis have been shown in rat and rabbit models after administration of HBOCs (24, 33). These in vivo effects of HBOCs on platelet function can be related to nitric oxide sequestration by the heme iron of HBOCs (31, 32) or to generation of platelet-reactive free radicals (2, 30). Direct consequences of these effects are vasoconstriction, impairment of microvascular function, and tissue and microvascular O2 consumption (16).
Experimental studies on the effects of HBOCs and non-O2-carrying plasma expanders administered in animal models yield conflicting results regarding how O2 delivery and tissue PO2 are affected by the presence of these compounds in the circulation (15). Studies using PBH to restore O2-carrying capacity in extreme hemodilution (39), where Hb concentration was 6.7 g/dl of blood (3.4 g Hb/dl from RBCs and 3.3 g Hb/dl from PBH), resulted in PO2 of 0.3 mmHg, and the number of capillaries perfused per unit of area [functional capillary density (FCD)] decreased to 37 ± 0.21% of baseline. Conversely, the same level of hematocrit (Hct) reduction achieved with dextran 70 to total Hb of 3.4 g Hb/dl from RBCs caused tissue PO2 to be 3.4 mmHg and FCD to be 53 ± 18% of baseline.
Standl et al. (36) subjected dogs to isovolemic hemodilution with 6% hetastarch, reducing their Hct to 10%, and then augmented Hb with PBH; they found that mean tissue PO2 measured with a needle electrode increased to 56 mmHg after transfusion (normal nontreated animals had tissue PO2 of 27 mmHg). This phenomenon was accompanied by a 59% increase (from baseline) in systemic O2 extraction. In another study, the same investigators further reduced systemic Hct to 5% before infusion of PBH and found a similar increase in tissue PO2 (36, 37).
The anomalous partition O2 reported by Tsai (39) was further analyzed by Tsai et al. (43), who found that O2 extraction in the PBH extreme hemodilution protocol was 25% higher than in dextran, a trend that corroborates the findings of Standl et al. (36, 37). However, tissue PO2 was significantly lower than at baseline or with dextran hemodilution.
In previous studies (36, 37), PBH was used in concentrations ranging from 1.5 to 7.2 g Hb/dl, which may overcome the negative effects of vasoconstriction on O2 delivery associated with use of this material, according to a theoretical model showing that free Hb in plasma facilitates off-loading of O2 from RBCs proposed by Federspiel (14). An additional mechanism that further facilitates O2 delivery from RBCs to tissue is facilitated diffusion (29). However, neither of these mechanisms explains the results of Tsai et al. (43), showing that a trend of increased O2 extraction leads to lower tissue PO2 in the hamster window chamber.
The possibility exists that some free molecular Hb formulations affect the rate of O2 consumption of the vessel wall (endothelium and smooth muscle) upon extravasation, as a consequence of toxic effects involving the formation of O2 free radicals as the molecule undergoes rapid oxidation in vivo (2). As these Hb molecules further diffuse into the tissue, these effects may ultimately extend to the parenchyma, because the interstitial fluid lacks the enzymatic protective mechanisms to maintain Hb in a reduced form. The high O2 consumption by the vessel wall has been established (16, 41, 43); therefore, elimination or incapacitation of this O2 sink would make more O2 available to the tissue, thus increasing its PO2.
The present study was designed to determine O2 delivery and extraction in the hamster window model in a condition where most of the O2 is carried by PBH introduced into the circulation by an 80% exchange transfusion. In these experiments, we measured the delivery and extraction of O2 in the microcirculation by RBCs and plasma Hb separately, after a one-step hemodilution to a Hct of 19%. Effects were studied at 1, 12, and 24 h after exchange transfusion to document the progression of tissue O2 distribution and consumption in relation to the half-life of the Hb solution.
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METHODS |
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2 days for recovery; then its chamber was assessed under the microscope for any signs of edema, bleeding, or unusual neovascularization. Barring these complications, the animal was anesthetized again with pentobarbital sodium. Arterial and venous catheters (PE-50) were implanted in the carotid artery and jugular vein. The catheters were filled with a heparinized saline solution (30 IU/ml) to ensure their patency at the time of the experiment. Catheters were tunneled under the skin and exteriorized at the dorsal side of the neck, where they were attached to the chamber frame with tape. The experiment was performed after 24 h but within 48 h after catheter implantation. Inclusion criteria. Animals were suitable for the experiments if 1) systemic parameters were within the normal range: heart rate (HR) >340 beats/min, mean arterial blood pressure (MAP) >80 mmHg, systemic Hct >45%, and arterial PO2 (PaO2) >50 mmHg; and 2) microscopic examination of the tissue in the chamber observed under x650 magnification revealed no signs of edema or bleeding.
Systemic parameters. MAP and HR were recorded continuously (model MP 150, Biopac System, Santa Barbara, CA), except during the actual blood exchange. Hct was measured from centrifuged arterial blood samples taken in heparinized capillary tubes (Readacrit Centrifuge, Clay Adams, Division of Becton-Dickinson, Parsippany, NJ). Hb content was determined spectrophotometrically from a single drop of blood (B-Hemoglobin, Hemocue, Stockholm, Sweden).
Blood chemistry and colloid osmotic pressure. Arterial blood was collected in heparinized glass capillaries (0.05 ml) and immediately analyzed for PaO2, arterial PCO2 (PaCO2), base excess, and pH (Blood Chemistry Analyzer 248, Bayer, Norwood, MA). Blood samples for colloid osmotic pressure (COP) measurements were withdrawn from the animal with a heparinized 3-ml syringe at the end of the experiment for immediate analysis. Blood samples were centrifuged, and COP in the plasma was measured using a membrane colloid osmometer (model 420, Wescor, Logan, UT). The osmometer was calibrated with a 5% albumin solution using a 30-kDa-cutoff membrane (Amicon, Danvers, MA) (45). Whole blood lactate concentration was measured from a 25-µl sample (model 1500 SPORT Lactate Analyzer, Yellow Springs Instrument, Yellow Springs, OH).
FCD. Functional capillaries, defined as capillary segments with RBC transit of at least a single RBC in a 30-s period, were assessed in 10 successive microscopic fields, totaling a region of 0.46 mm2. The fields were observed systematically by displacement of the microscopic field of view by a field width in 10 successive steps in the lateral direction (relative to the observer). Each step was viewed on the video monitor and was 200 µm long when referred to the tissue. The first field was chosen by a distinctive anatomic landmark (i.e., large vascular bifurcation) to easily and quickly reestablish the same fields and vessels at each observation time point. Each field had two to five capillary segments with RBC flow. FCD (cm–1), i.e., total length of RBC-perfused capillaries ÷ area of the microscopic field of view, was evaluated by measuring and adding the length of capillaries that had RBC transit in the field of view. The relative change in FCD from baseline after each intervention is indicative of the extent of capillary perfusion.
Microhemodynamics.
Arteriolar and venular blood flow velocities were measured online using the photodiode cross-correlation method (19) (Photo Diode/Velocity Tracker 102B, Vista Electronics, San Diego, CA). The measured centerline velocity (V) was corrected according to vessel size to obtain the mean RBC velocity (27). A video image-shearing method was used to measure vessel diameter (D) (20). Blood flow (
) was calculated from the measured values as = V x 
(D/2)2. Changes in arteriolar and venular diameter from baseline were used as indicators of a change in vascular tone.
Acute isovolemic hemodilution. Progressive hemodilution to a final systemic Hct of 19% was accomplished with a single-step isovolemic exchange. Briefly, the volume of the exchange transfusion was calculated as a percentage of the blood volume, estimated as 7% of body weight. The acute anemic state was induced by lowering systemic Hct by 80% with a progressive isovolemic hemodilution using PBH: 13.1% PBH solution in a modified lactated Ringer solution (Oxyglobin). Table 1 lists the physical characteristics of the test solution.
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Experimental groups. Animals were randomly divided into three experimental groups according to the time after exchange transfusion in which microvascular O2 distribution and consumption were determined. Corresponding O2 measurements were performed at 1, 12, and 24 h after exchange transfusion. A group that did not undergo the hemodilution protocol served as baseline O2 distribution for this study.
Experimental setup and procedure. The unanesthetized animals were placed in a restraining tube with a longitudinal slit from which the window chamber protruded. The animals were given 30 min to adjust to the tube environment before baseline systemic parameters (MAP, HR, blood gases, and Hct) were measured. The conscious animal in the tube was then fixed to the microscopic stage of a transillumination intravital microscope (model BX51WI, Olympus, New Hyde Park, NY). The tissue image was projected onto a charge-coupled device camera (COHU 4815) connected to a videocassette recorder (model AG-7355, JVC) and viewed on a monitor. Measurements were carried out using a x40 (LUMPFL-WIR, numerical aperture 0.8, Olympus) water-immersion objective. Detection of RBC passage was enhanced by increasing the contrast between RBCs and tissue with a BG12 (420-nm) band-pass filter.
Fields of observation and vessels were chosen for study at locations in the tissue where the vessels were in sharp focus. Detailed maps were made of the chamber vasculature to record the vessel location and ensure that the same microvessels were studied throughout the experiment. Measurements were performed after exchange and the ensuing stabilization period. Blood samples for analysis of COP were withdrawn for each group of animals studied at each specific time after completion of all systemic and microhemodynamic measurements at that time.
Measurement of cell-free MetHb.
Plasma Hb was collected from microhematocrit tubes 2 min after centrifugation, and 50 µl of the RBC-free solution were diluted in 20 vol of deionized water. The MetHb level was determined according to previous methods (48), difference in absorbance between 577 and 630 nm, adjusted for the respective extinction coefficients of oxyhemoglobin and MetHb. In this cell-free Hb, there was a background of absorption at 630 nm due to turbidity. This background level was used to correct each determination of MetHb, and the results were expressed as percent increase in MetHb relative to total Hb. Calibration was ensured using standard levels of 5.2, 2.6, and 1.2% MetHb (RNA Medical, CO-Oximeter Control, Bayer Diagnostics, Medfield, MA). In normal conditions, the concentration of cell-free Hb in the hamster is <0.08 g Hb/dl, and cell-free MetHb levels are almost impossible to detect.
Hemoglobin O2 saturation. O2 saturation of RBC Hb and PBH was investigated by deoxygenation of O2-equilibrated oxyhemoglobin in Hemox buffer (pH 7.4) at 37.6°C using a Hemox analyzer (TCS Scientific, New Hope, PA), which measures the O2 pressure with a Clark-type O2 electrode (Yellow Springs Instrument) and simultaneously calculates the Hb saturation via a dual-wavelength spectrophotometer. PO2 at which Hb is half-saturated was obtained directly from the O2 equilibrium curves (OECs). The OEC for hamster RBCs was determined from freshly collected blood, and the OEC for PBH was measured from fresh material. The Hemox analyzer program assumes that Hb is 100% saturated at 150 mmHg; however, PBH is only 72.1% saturated at 150 mmHg; therefore, saturation data were corrected for this discrepancy (22).
Microvascular PO2 distribution. High-resolution microvascular PO2 measurements were made using phosphorescence quenching microscopy (38). This method for measuring O2 levels is based on the O2-dependent quenching of phosphorescence emitted by albumin-bound metalloporphyrin complex after pulsed light excitation. Phosphorescence microscopy is not dependent on the level of dye within the tissue, and the decay time is inversely proportional to PO2. The phosphorescence decay curves were converted to PO2 using a fluorescence decay curve fitter (model 802, Vista Electronics, Ramona, CA) (21). This technique has been used in this animal preparation and others for intravascular and extravascular PO2 measurements, inasmuch as albumin exchange between plasma and tissue allows for sufficient concentrations of albumin-bound dye within the interstitium to achieve an adequate signal-to-noise ratio. Animals received a slow intravenous injection of palladium-meso-tetra(4-carboxyphenyl)porphyrin (Porphyrin Products, Logan, UT; 15 mg/kg body wt at 10.1 mg/ml), and the dye was allowed to circulate for 20 min before PO2 measurements.
In our system, intravascular measurements are made by placing an optical rectangular window within the vessel of interest, with the longest side of the rectangular slit positioned parallel to the vessel wall. Measurements are made by placing a rectangular optically in the window region of 5 x 40 µm longitudinally within the vessel. Tissue PO2 was measured in regions without large vessels within intercapillary spaces with an optical window size of 10 x 10 µm. Thus the exact location of the PO2 measurements is known: intravascular in an arteriole or venule or in the interstitial tissue (41). The phosphorescence decay due to quenching at a specific PO2 yields a single decay constant, and in vitro calibration has been demonstrated to be valid for in vivo measurements. Intravascular and perivascular PO2 was measured in the arterioles studied, and intravascular PO2 was measured in venules. Interstitial tissue PO2 was measured within the interstitium distant from visible underlying and adjacent vessels.
Tissue O2 delivery and extraction.
The microvascular methodology used in our studies allows a detailed analysis of O2 supply in the tissue. O2 delivery, defined as amount of O2 per unit time delivered by the arterioles to the microcirculation relative to control, and O2 extraction, defined as the amount of O2 released by blood in the microcirculation per unit time relative to control, are calculated as follows
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 | (2) |
is O2-carrying capacity of Hb at 100% saturation or 1.34 ml O2/g Hb, SA% is arteriolar O2 saturation of RBCs, 
A% is arteriolar O2 saturation of PBH, the subscript A–V indicates the difference between arterioles and venules, (1 – Hct) is the fractional plasma volume and converts the equation from per deciliter of plasma to per deciliter of blood, 
is solubility of O2 in plasma equal to 3.14 x 10–3 ml O2·dl plasma–1·mmHg–1, PO2 A is arteriolar PO2, PO2 A-V is arteriolar-venular difference in PO2, and is microvascular flow for each microvessel as percentage of baseline. OECs were determined as described above (see Hb O2 saturation).
Longitudinal O2 exit from arterioles.
The analysis of O2 supply to the tissue allows us to calculate the rate of O2 exit from arterioles to the tissue per unit vessel length as follows
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SA% is the change in O2 saturation of RBCs along the vessel, A% is the change in O2 saturation of PBH along the vessel, PO2 A is the change in PO2 along the vessel, is the microvascular flow for each microvessel as a percentage of baseline, and l is the distance along the vessel where blood PO2 is measured (0.5–1.0 mm). Data analysis. Values are means ± SD unless otherwise noted. Data within each group were analyzed using ANOVA for repeated measurements. When appropriate, post hoc analyses were performed with Tukey's multiple comparison test. All data are presented as absolute values and ratios relative to baseline values. A ratio of 1.0 signifies no change from baseline; lower and higher ratios indicate changes proportionally higher and lower than baseline. The same vessels and functional capillary fields were followed, so that direct comparisons with their baseline levels could be performed, allowing for more robust statistics for small sample populations. All statistics were calculated using GraphPad Prism version 4.01 (GraphPad Software, San Diego, CA). Changes were considered statistically significant if P < 0.05.
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RESULTS |
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Hematological changes. The blood exchange transfusion resulted in a final Hct of 19.5 ± 0.6% after 1 h for all experiments. The PBH exchange transfusions resulted in a plasma Hb of 6.3 ± 0.4 g/dl, which increased total Hb in the blood (RBCs + Hb dissolved in plasma) to 11.7 ± 0.4 g/dl at 1 h after exchange transfusion. O2-carrying capacity, i.e., the total Hb content of blood, decreased progressively: 10.2 ± 0.4 g/dl (plasma Hb = 3.6 ± 0.3 g/dl) at 12 h and 9.1 ± 0.4 g/dl (plasma Hb = 2.8 ± 0.3 g/dl) at 24 h.
COP. All animals converged to COP of 17.9 ± 0.8 mmHg, the value for normal blood for this species (5, 6), at 1 h after exchange. This result indicates that introduction of the bulk solution into the circulation causes autotransfusion, which dilutes the administered material, leading to colligative properties identical to those at baseline, regardless of the relative values of COP in the original solution. At 12 and 24 h after exchange, COP was 17.7 ± 0.6 and 17.2 ± 0.7 mmHg, respectively.
Systemic and blood gas parameters. Systemic and blood gas parameters are presented in Table 2. The comparatively low PaO2 and high PaCO2 of these animals signify a consequence of their adaptation to a fossorial environment. Although PaCO2 remained slightly decreased from baseline throughout the experimental procedure, the change was not statistically significant, and there was no evidence of change in the breathing pattern.
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There was a statistical increase in PO2 at 1 h after exchange with PBH. However, it was not different from baseline at 12 and 24 h. PaCO2 was reduced from baseline after exchange transfusion. Blood pH was not statistically changed. Base excess was positive and continued to decrease from baseline after exchange. Lactate levels showed an increasing trend at 1, 12, and 24 h after exchange but were not significantly different from baseline.
Microhemodynamics. At 1 h after exchange, arteriolar and venular diameters were statistically different from baseline: 0.78 ± 0.11 and 0.86 ± 0.10, respectively. Arteriolar diameter did not fully recover at 12 h (0.88 ± 0.10), but venular diameter did recover. At 24 h, arteriolar and venular diameters were not different from baseline. Arteriolar and venular flow velocities were significantly decreased from baseline: 0.70 ± 0.23 and 0.76 ± 0.22, respectively, at 1 h. At 12 and 24 h, arteriolar and venular flow velocities recovered. Combining diameter and flow velocity data allowed us to calculate arteriolar and venular flows (Fig. 1), indicating that, at 1 h after exchange, arteriolar and venular flows were reduced from baseline: 0.43 ± 0.28 and 0.55 ± 0.29, respectively. Arteriolar flow was significantly lower at 12 h after exchange (0.79 ± 0.34 of baseline) and recovered after 24 h. Venular flow was not different at 12 and 24 h after exchange.
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Microvascular O2 distribution. Arteriolar PO2 after exchange transfusion with PBH was not significantly different from baseline at 1, 12, and 24 h after exchange: 52 ± 6 mmHg (n = 20) at baseline, 50 ± 4 mmHg (n = 29) at 1 h, 53 ± 5 mmHg (n = 27) at 12 h, and 55 ± 4 mmHg (n = 28) at 24 h. Venular PO2 was maintained at 1 h after exchange (33 ± 4 mmHg, n = 28) and decreased at 12 and 24 h [21 ± 4 mmHg (n = 28) and 19 ± 3 mmHg (n = 26), respectively]. Venular PO2 after 12 and 24 h was significantly lower than baseline: 33 ± 5 mmHg (n = 20; Fig. 2).
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Arteriolar wall gradients were determined from the difference between intravascular and perivascular PO2 measurements across the vessel walls. This parameter has been shown to be directly related to the rate of O2 consumption of the vessel wall (16, 41). The vessel wall gradient in arterioles in baseline conditions was 16 ± 4 mmHg (n = 10). This value decreased to 9 ± 3 mmHg (n = 10) at 1 h and returned to 16 ± 6 mmHg (n = 10) at 12 h and 18 ± 6 mmHg (n = 10) at 24 h.
Cell-free MetHb. MetHb in the PBH solution before injection was 2 ± 1% (n = 12). At 1 h after exchange, oxidation of the cell-free Hb increased significantly, and MetHb was 9 ± 2% (n = 6). At 12 h after exchange transfusion, MetHb decreased to 4 ± 1% (n = 6) and was 3 ± 1% (n = 5) at 24 h after exchange transfusion.
Tissue O2 delivery and extraction. Calculations of O2 delivery (mean ± SD, ml O2/dl blood–1) showed the lowest O2 delivery and extraction at 1 h after exchange: delivery = 2.9 ± 0.6 ml O2/dl blood, extraction = 0.9 ± 0.3 ml O2/dl blood, and extraction ratio = 31%. At 12 and 24 h, O2 delivery and extraction increased: at 12 h, delivery = 4.2 ± 0.8 ml O2/dl blood, extraction = 2.7 ± 0.6 ml O2/dl blood, and extraction ratio = 66%; at 24 h, delivery = 4.5 ± 0.9 ml O2/dl blood, extraction = 3.3 ± 0.7 ml O2/dl blood, and extraction ratio = 76% (Fig. 3). Baseline O2 delivery and extraction were 7.3 ± 1.1 and 3.1 ± 0.8 ml O2/dl blood, respectively, and extraction ratio was 42%.
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DISCUSSION |
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The changes in O2 delivery were paralleled by changes in the amount of O2 released by arterioles to the tissue, as calculated from measurements of the longitudinal and transmural O2 gradients. At 1 h, O2 release from arterioles was 10% less than baseline (but not statistically significantly different) and was significantly increased from baseline at 12 and 24 h. O2 exit from the arterioles is determined by the transmural O2 gradients and was significantly reduced at 1 h. The transmural O2 gradient progressively recovered to normal values at 24 h.
Our findings are consistent with the possibility that the presence of PBH in the circulation causes a significant decrease in O2 consumption, which results in more O2 available in the tissue and, therefore, a higher tissue PO2. The latter could be, in part, due to the low affinity of PBH, which favors O2 unloading, as shown by Page et al. (34); however, other factors must be present, because the total amount of O2 delivered and consumed is decreased. The parameter that characterizes O2 consumption by the microvascular wall is the O2 gradient measured across this structure. In the present study, the vessel wall gradient decreased significantly at 1 h, indicating that vessel wall O2 consumption was significantly depressed. Therefore, if vessel wall O2 consumption is a significant O2 sink, as shown by some studies (16, 41, 49), and this is depressed, as evidenced by the decrease in vessel wall gradient, it follows that more O2 would be available to the tissue, resulting in higher tissue PO2. These phenomena may be exacerbated by a concomitant decrease in tissue O2 consumption. If only vessel wall O2 metabolism is affected by the presence of molecular Hb in plasma, the O2 need of the tissue, as a whole, would be lowered to 70% of baseline. However, our results show that O2 released into the tissue is reduced to 30% of baseline, indicating a reduction of O2 consumption by an additional compartment, i.e., the parenchyma.
Endothelial cells are especially susceptible to Hb-induced toxicity because of their proximity to circulating blood. Enzymes such as xanthine oxidase, NADH-NADPH oxidase, and myeloperoxidase may play prominent roles in driving the oxidative reactions of Hb in the vasculature (7). Oxidative stress can induce a wide array of cellular responses, including growth stimulation, temporary arrest and adaptation, permanent arrest, apoptosis, and necrosis, depending on its strength or duration (11).
Even though PBH caused significantly lower levels of tissue O2 consumption at 1 h after exchange, we found no evidence for global tissue hypoxia compared with conditions before exchange transfusion. Arterial lactate values were increased, but in a range not representative of physiological changes, and acid-base balance continued to be positive. PBH is a vasoactive material, an effect that is attributed to nitric oxide scavenging by the free molecular Hb, evidenced by vasoconstriction of arteriolar and venular vessels, and the concomitant decrease in blood flow. Therefore, maintenance (and, in our case, increase) of tissue PO2 should have correlated with an increase of extraction, i.e., O2 release by the microcirculation into the tissue. However, this was not evident, in view of the finding of significantly reduced rates of arteriolar O2 exit.
Our protocol was designed to determine whether maintaining blood O2-carrying capacity with use of PBH normalizes O2 delivery to the tissue and microvascular function while maintaining Hct at 19% and total Hb at 11.7 g/dl. This Hb level was well above that of 5.4 g Hb/dl used by Tsai et al. (41) in a similar normovolemic hemodilution study in the same model with 70-kDa dextran, where O2 delivery and tissue PO2 (20.4 mmHg) attained normal values. Therefore, the decrease in O2 delivery is not attributable to a deficit in O2-carrying capacity, because the same amount of Hb in RBCs, in the absence of PBH, provides for normal tissue O2 delivery and tissue PO2. There are, however, indications in the literature that, in previous studies in animal models of hemorrhagic shock or isovolemic hemodilution, PBH infusion failed to increase arterial O2 content (12, 18) or systemic O2 transport (23, 44). Conversely, administration of large quantities of PBH in studies of massive blood loss in dogs and sheep resulted in excellent intravascular volume expansion and increased O2-carrying capacity with no evidence of toxicity (4, 25, 28). Furthermore, Standl et al. (36, 37) showed that use of PBH in an extreme hemodilution protocol when RBCs were substituted with PBH as an O2 carrier increased tissue PO2.
These discrepancies can be in part explained by our findings. It would appear that the initial introduction of PBH into the circulation has a direct effect on microvascular and tissue O2 metabolism, which is significantly reduced. This reduction is notable, because in some instances, such as the studies of Standl et al. (36, 37) and the data presented in this study, tissue PO2 was increased, even though O2 delivery decreased as a result of vasoconstriction. These effects subside in time; therefore, analysis of the O2-carrying capacity and delivery of HBOCs with characteristics similar to PBH are dependent in part on the time at which the organism is studied.
The oxidation (autoxidation) of Hb to a nonfunctional ferric (MetHb) form is a factor in the use of Hb as an O2-carrying product. This uncontrolled and spontaneous oxidation of ferrous iron compromises the O2-carrying function of Hb and, in some instances, the safety of infused HBOCs. The question of how much MetHb is too much was addressed in a study based on a rat model subjected to a 30% exchange transfusion of conjugated Hb with polyethylene glycol (PEG-Hb) (26). It was estimated that >10% MetHb can significantly decrease the ability of this Hb to oxygenate tissues. Hb autoxidizes naturally, a process that is controlled within the RBCs by the MetHb reductase system. When this enzyme is not present, MetHb superoxide ions are formed, which in further reactions with heme produce H2O2, a highly reactive species that causes cellular damage, including growth arrest, cellular injury, apoptosis, and necrosis in endothelial cell cultures. Furthermore, iron release after reactions of heme with H2O2 acts as an oxidant capable of damaging lipids, nucleic acids, and amino acids. The biochemical basis for this potential toxicity has been extensively reviewed by Alayash (3).
The rate of MetHb formation in the present study was somewhat lower than that of other similar products, e.g., -cross-linked Hb, which is reported to have a higher rate of MetHb formation. MetHb toxicity is related to the location of heme within the tissue, which is directly related to the molecular dimensions of the Hb O2 carrier. Thus PBH should have fewer side effects than -cross-linked Hb but ranks behind PEG-conjugated Hb and vesicle-encapsulated Hb. In this context, one may speculate that toxicity results from the combination of the rate of MetHb formation and the facility of a given molecule to extravasate, which is dependent on molecular dimension. Thus PBH should result in fewer side effects than -Hb but would rate behind PEG-conjugated Hb and vesicle-encapsulated Hb.
A possible mechanism for the decrease in O2 metabolism of the vessel wall and tissue may be related to the type of chemistry introduced to stabilize and functionally modify the Hb. The chemical modifications can indeed determine the vulnerability of Hb to undergo oxidative self-destructive side reactions and not only modify key amino acids (i.e., amino acids involved in Bohr and chloride effects and CO2 binding) but also have far-reaching consequences, including distortion of Hb geometry and loss of its integrity (50). At the cellular level, differences in the rates of autoxidation and oxidative side reactions not only determine the ability of Hb to induce injury but can also determine its ability to deliver O2. Different HBOCs can differentially modulate key cell-signaling pathways and other important physiological mediators, including alteration of mitochondrial function. Yeh and Alayash (50) showed that HBOCs cross talk with hypoxia inducible factor-1 in endothelial cells subjected to normoxia and more so in hypoxia. Changes in the expression of hypoxia inducible factor-1 and other important signaling proteins are clearly dependent on the O2-carrying properties and redox state of Hb (50).
The present study shows that O2 metabolism is significantly reduced when RBCs are replaced by PBH as an O2 carrier. A speculative question is whether the physiological significance of this finding is positive or negative. It may be argued that if this HBOC were used in resuscitation of a traumatized subject, the depression of O2 metabolism may be an aggravating factor complicating recovery. However, in a condition of limited O2 supply, such as in resuscitation from hemorrhagic shock, a decrease in O2 requirements by the microcirculation and the parenchyma would lower O2 requirements and provide for an increased tissue PO2, as shown by our findings. A concomitant positive effect of this outcome is that elevation of tissue PO2 due to lowered rate of O2 metabolism should reduce formation of hypoxia-generated O2 free radicals, a factor that may explain the virtually complete recovery of our model at 24 h.
In conclusion, exchange of RBC-based blood O2-carrying capacity with PBH leads to a significant decrease in O2 delivery to the microcirculation of the hamster window chamber model. Paradoxically, this effect results in a significant increase in tissue PO2. This finding suggests that the O2 requirements of the microcirculation and the tissue are reduced as a result of a direct effect of molecular Hb on the vessel wall and the tissue. These effects are temporary, are most prominent at 1 h after exchange, and are reversible, suggesting that infusion of this material in a normal organism may protect the tissue from the effects of hypoxia.
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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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A. S. Golub, M. C. Barker, and R. N. Pittman PO2 profiles near arterioles and tissue oxygen consumption in rat mesentery Am J Physiol Heart Circ Physiol, August 1, 2007; 293(2): H1097 - H1106. [Abstract] [Full Text] [PDF]
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P. Cabrales and A. G. Tsai Plasma viscosity regulates systemic and microvascular perfusion during acute extreme anemic conditions Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2445 - H2452. [Abstract] [Full Text] [PDF]
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P. Cabrales, J. Martini, M. Intaglietta, and A. G. Tsai Blood viscosity maintains microvascular conditions during normovolemic anemia independent of blood oxygen-carrying capacity Am J Physiol Heart Circ Physiol, August 1, 2006; 291(2): H581 - H590. [Abstract] [Full Text] [PDF]
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P. Cabrales, A. G. Tsai, R. M. Winslow, and M. Intaglietta Extreme hemodilution with PEG-hemoglobin vs. PEG-albumin Am J Physiol Heart Circ Physiol, December 1, 2005; 289(6): H2392 - H2400. [Abstract] [Full Text] [PDF]
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P < 0.05 vs. baseline. Diameters [µm, means ± SD of number of vessels studied (n)] were as follows: baseline 56.1 ± 8.5 (n = 104) for arterioles and 62.5 ± 12.4 (n = 102) for venules at baseline, 48.2 ± 12.8 (n = 84) for arterioles and 54.2 ± 11.5 (n = 82) for venules at 1 h after exchange, 51.2 ± 14.1 (n = 55) for arterioles and 59.6 ± 15.2 (n = 54) for venules at 12 h after exchange, and 57.2 ± 13.8 (n = 28) for arterioles and 60.5 ± 12.7 (n = 26) for venules at 24 h after exchange. Red blood cell (RBC) velocities (mm/s, means ± SD) were as follows: 4.6 ± 2.2 for arterioles and 1.4 ± 0.5 for venules at baseline, 3.1 ± 1.5 for arterioles and 1.0 ± 0.8 for venules at 1 h after exchange, 4.0 ± 1.5 for arterioles and 1.2 ± 0.9 for venules at 12 h after exchange, and 4.2 ± 1.8 for arterioles and 1.4 ± 0.8 for venules at 24 h after exchange. Flows (nl/s, means ± SE) were as follows: 11.4 ± 1.8 for arterioles and 4.1 ± 1.0 for venules at baseline, 5.1 ± 1.3 for arterioles and 2.5 ± 1.9 for venules at 1 h after exchange, 9.0 ± 1.6 for arterioles and 3.8 ± 1.8 for venules at 12 h after exchange, and 10.2 ± 2.9 for arterioles and 4.4 ± 1.9 for venules at 24 h after exchange.
