Hemoglobin (Hb)-based O2 carriers (HBOCs) constitute a class of therapeutic agents designed to correct the O2 deficit under conditions of anemia and traumatic blood loss. The O2 transport capacity of ultrahigh-molecular-weight bovine Hb polymers (PolybHb), polymerized in the tense (T) state and relaxed (R) state, were investigated in the hamster chamber window model using microvascular measurements to determine O2 delivery during extreme anemia. The anemic state was induced by hemodilution with a plasma expander (70-kDa dextran). After an initial moderate hemodilution to 18% hematocrit, animals were randomly assigned to exchange transfusion groups based on the type of PolybHb solution used (namely, T-state PolybHb and R-state PolybHb groups). Measurements of systemic parameters, microvascular hemodynamics, capillary perfusion, and intravascular and tissue O2 levels were performed at 11% hematocrit. Both PolybHbs were infused at 10 g/dl, and their viscosities were higher than nondiluted blood. Restitution of the O2 carrying capacity with T-state PolybHb exhibited lower arterial pressure and higher functional capillary density compared with R-state PolybHb. Central arterial O2 tensions increased significantly for R-state PolybHb compared with T-state PolybHb; conversely, microvascular O2 tensions were higher for T-state PolybHb compared with R-state PolybHb. The increased tissue Po2 attained with T-state PolybHb results from the larger amount of O2 released from the PolybHb and maintenance of macrovascular and microvascular hemodynamics compared with R-state PolybHb. These results suggest that the extreme high O2 affinity of R-state PolybHb prevented O2 bound to PolybHb from been used by the tissues. The results presented here show that T-state PolybHb, a high-viscosity O2 carrier, is a quintessential example of an appropriately engineered O2 carrying solution, which preserves vascular mechanical stimuli (shear stress) lost during anemic conditions and reinstates oxygenation, without the hypertensive or vasoconstriction responses observed in previous generations of HBOCs.
- hemoglobin-based oxygen carrier
- oxygen carrying capacity
- blood substitute
- polymerized hemoglobin
- exchange transfusion
- tissue oxygen
- hemoglobin oxygen affinity
the development of hemoglobin (Hb)-based O2 carriers (HBOCs) represents a concentrated effort to engineer a replacement for red blood cells (RBCs). Although earlier attempts to develop HBOCs were imaginative and stimulated great research effort, few came to fruition, since satisfactory performance, as well as scientific knowledge about how O2 is transported and extracted from the blood, had not been presented with sufficient evidence. The crisis of infectious disease transmission (in the 1980s) generated renewed interest in the HBOC research field, since the use of RBC replacement solutions allows the adverse consequences of transfusions to be avoided (34). The potential transmission of human immunodeficiency virus stimulated the development of a new generation of potentially useful solutions that could function as RBC substitutes. However, although many of the RBC substitutes showed promise in the initial preclinical testing phase, few survived the rigors of clinical regulatory oversight and safety considerations.
Once the challenges of HBOCs, i.e., the molecular stability of Hb and appropriate O2 affinity, were partially addressed, some compounds entered clinical testing (16, 24). Through a variety of molecular modifications, more stable Hb polymers were created that effectively minimized renal clearance of the protein (33). Efforts to create modified Hb through recombinant technology were undertaken, with varying degrees of success (33). Unfortunately, these efforts proved to be prohibitively expensive. Scaling the process up to produce the quantities required to meet the anticipated demands were feared to exceed the costs that could be sustained in the eventual marketplace. Nevertheless, this recombinant technology remains an excellent approach for future research.
Current HBOCs result in vasoconstriction, which is evident as a rapid rise in peripheral vascular resistance and blood pressure, commonly associated with bradycardia, decreased cardiac output, and reduced perfusion to vital organs (15, 28, 36). Vasoconstriction is currently perceived to be the critical barrier hampering HBOC development (1, 41). Vasoconstriction appears to be directly linked to nitric oxide (NO) scavenging, to an oversupply of O2 to the blood vessel wall, and extravasation (19). Regardless of the exact mechanism for vasoconstriction, the presence of acellular Hb in solution enables these various potential mechanisms of vasoconstriction (29).
Polymerization of Hb can be used to yield larger-sized Hb particles that can interact strongly with each other, especially when the polymerized Hb solution is highly concentrated. Hence, polymerization of bovine Hb (bHb) yields high-molecular-weight (MW) fractions that display an increase in viscosity with increasing cross-link density and low colloidal osmotic pressure (COP). At higher cross-link densities (40:1 and 50:1), the bHb polymers (PolybHbs) have the majority of intramolecular cross-linking sites saturated, which facilitates subsequent intermolecular cross-linking between adjacent Hb tetramers in solution. The molecular size of PolybHb has a direct impact on its proximity to the vascular endothelium and its molecular diffusivity, theoretically decreasing NO scavenging and vessel wall O2 oversupply. These properties should prevent the induction of vasoconstriction.
A previous study (5) published by our group suggested a direct correlation between the hypertensive response and the constriction of resistance arterioles after the hypervolemic infusion of PolybHb solutions with MWs below 500 kDa. On the other hand, PolybHb solutions with high cross-link densities and MWs above 500 kDa increased the plasma O2 carrying capacity and plasma viscosity without inducing vasoconstriction or microcirculatory disturbances (5). Under extreme anemic conditions, the O2 delivered becomes insufficient to meet the O2 demand, and high-MW PolybHb solutions could be a potential alternative to allogeneic blood transfusion. This study was designed to understand how the quaternary state, oxygenated [relaxed (R)] and deoxygenated [tense (T)], of high-MW PolybHbs during polymerization affects their O2 transport and O2 delivery characteristics during extreme anemic conditions. The extreme anemic state in the hamster window chamber model is attained at a hematocrit (Hct) of 11% and is a powerful tool to test the efficacy of HBOCs to maintain systemic and microvascular function as well as oxygenation. This Hct is below the threshold at which the animal organism becomes O2 supply limited and magnifies the effects of transfused HBOCs.
Glutaraldehyde (70%), NaCl, KCl, NaOH, Na2S2O4, NaCl (USP), KCl (USP), CaCl2·2H2O (USP), NaOH (NF), sodium lactate (USP), N-acetyl-l-cysteine (USP), NaCNBH3, and NaBH4 were purchased from Sigma-Aldrich (Atlanta, GA). Sephadex G-25 resin was purchased from GE Healthcare (Piscataway, NJ). KCN, KFe(CN)6, and all other chemicals were purchased from Fisher Scientific (Pittsburgh, PA).
Fresh bovine blood collected in 3.8% sodium citrate solution at a final concentration of 90:10 (vol/vol) (bovine blood-sodium citrate solution) was purchased from Quad Five (Ryegate, MO). bHb was purified from lysed bovine RBCs (bRBCs) via tangential flow filtration (12, 31). bRBCs were initially washed three times with 3 volumes of isotonic saline solution (0.9%) at 4°C. bRBCs were subsequently lysed on ice with 2 volumes of hypotonic 3.75 mM phosphate buffer at pH 7.4 for 1 h. The lysate was then filtered through a glass chromatography column packed with glass wool to remove the majority of cell debris. Clarified bRBC lysate was then passed through 50-nm and 500-kDa hollow fiber cartridges (Spectrum Labs, Rancho Dominguez, CA) to remove additional cell debris and impurity proteins. Purified bHb was collected and concentrated on a 100-kDa hollow fiber cartridge (Spectrum Labs) to yield the precursor material for PolybHb synthesis.
Polymerization of bHb.
T-state PolybHb was synthesized according to previously described methods (5, 12). To generate fully deoxygenated (T-state) bHb, 30 g of purified bHb were diluted with phosphate buffer (20 mM, pH 8.0) to yield 1,200 ml of bHb solution. The bHb solution was kept in a 2-liter glass bottle that was connected to a vacuum manifold and submerged in an ice water bath. The bHb solution was then subjected to several cycles of vacuum and argon purging to remove the majority of O2 from the aqueous solution and the gas headspace of the bottle. After 4 h of vacuum and argon cycling, Na2S2O4 solution (1.5 mg/ml) was titrated into the bHb solution while the Po2 of the solution was simultaneously measured using a RapidLab 248 (Siemens, Malvern, PA) blood gas analyzer until the Po2 of the bHb solution attained a value of 0 mmHg. At this point, an additional 30 ml of 1.5 mg/ml Na2S2O4 solution were added to the T-state bHb solution to maintain the Po2 at 0 mmHg during and after the polymerization reaction. A 30-ml syringe with a luer lock was used to titrate glutaraldehyde preequilibrated with argon in the sealed glass bottle under continuous stirring. For this polymerization reaction, the molar ratio of glutaraldehyde to bHb was 50:1.
Oxygenated (R-state) bHb was prepared in a similar manner to T-state bHb using the same vacuum manifold system. In this case, 1,500 ml of 0.3 mmol/l bHb solution were saturated with pure O2 in the vacuum manifold system for 2 h in an ice water bath. The Po2 measured was well over the 749-mmHg measurement range of the RapidLab 248 blood gas analyzer. Subsequently, a 30-ml syringe with a luer lock was used to titrate glutaraldehyde in the sealed glass bottle under continuous stirring. For this polymerization reaction, the molar ratio of glutaraldehyde to bHb was 40:1.
The resulting T-state or R-state bHb solutions were then allowed to react with glutaraldehyde at 37°C in a water bath for 2 h, which was stirred and equilibrated with either pure argon (50:1 T-state bHb) or O2 (40:1 R-state bHb). At the end of this period, for the 50:1 T-state bHb solution, 20 ml of 2 M NaBH4 in phosphate buffer (20 ml, pH 8.0) were injected into the glass bottle to quench the polymerization reaction. For the 40:1 R-state bHb solution, 5 ml of 8 M NaCNBH3 in phosphate buffer (20 ml, pH 8.0) were injected into the glass bottle to reduce the Schiff base and reduce the methemoglobin (metHb) level. The PolybHb solution was continuously stirred for 30 min in an ice water bath. Subsequently, 20 ml of 2 M NaBH4 in phosphate buffer (20 ml, pH 8.0) was injected into the glass bottle to quench the polymerization reaction. The Po2 of the bHb solution before polymerization, after polymerization, and after quenching with NaBH4 was measured using a RapidLab 248 blood gas analyzer (Siemens). All reactions were repeated in triplicate.
Separation of the PolybHb solution.
Initially, each PolybHb solution was clarified by passing it through a glass chromatography column packed with glass wool to remove large particles. The glass wool was previously autoclaved at 250°C for 30 min (39) before being used to clarify the PolybHb solution while all tubing, glassware, and plasticware were immersed in 1 M NaOH solution for >6 h to degrade any endotoxin present, followed by a thorough rinse with HPLC grade water (the mean conductivity value being 18.2 × 106 Ω·cm). The clarified PolybHb solution was then separated into two distinct MW fractions with a 500-kDa hollow fiber cartridge (Spectrum Labs). The retentate mostly contained PolybHb molecules that were larger than 500 kDa. This fraction was used for the experiments described in this study.
Buffer exchange of PolybHb solution.
After polymerization, the PolybHb solution was suspended in phosphate buffer along with reduced glutaraldehyde and excess NaBH4 (and NaCNBH3 for R-state PolybHb). Glutaraldehyde, NaCNBH3, and NaBH4 are cytotoxic (17); therefore, the PolybHb solution was buffer exchanged with a modified lactated Ringer solution [115 mmol/l NaCl (USP), 4 mmol/l KCl (USP), 1.4 mmol/l CaCl2·2H2O (USP), 13 mmol/l NaOH (NF), 27 mmol/l sodium lactate (USP), and 2 g/l N-acetyl-l-cysteine (USP)]. The buffer exchange was conducted using an äKTA Explorer 100 system controlled by Unicorn 5.1 software (GE Healthcare). An XK 50/30 (length: 300 mm, inner diameter: 50 mm) column (GE Healthcare) was packed with 500 ml of Sephadex G-25 medium resin at room temperature. The column was balanced with modified lactated Ringer solution at a flow rate of 8 ml/min, and the PolybHb solution was injected into the XK 50/30 column via a superloop column (50 ml, GE Healthcare) at a flow rate of 5 ml/min. The sample (100 ml) was injected at a time and then eluted with modified lactated Ringer solution. The protein concentration was detected at a wavelength of 280 nm while the salt concentration was monitored with a conductivity detector. During the buffer exchange process, the ultraviolet signal increased as PolybHb eluted from the column, whereas the conductivity decreased when reduced glutaraldehyde and NaBH4 (and NaCNBH3 for R-state PolybHb) eluted from the column. The buffer-exchanged PolybHb solution was collected as the ultraviolet signal increased but before the conductivity signal decreased. The PolybHb fraction was concentrated with a 100-kDa hollow fiber cartridge (Spectrum Labs).
MetHb level and protein concentration of PolybHb.
The metHb level of PolybHb solutions was measured via the cyanomethemoglobin method (11, 32). Total protein concentration was measured using the Bradford method (3) using the Coomassie Plus protein assay kit (Pierce Biotechnology, Rockford, IL).
Size exclusion chromatography coupled with multiangle static light scattering.
The absolute MW distribution of bHb/PolybHb solutions was measured using a size exclusion chromatography column (Ultrahydrogel linear column, 10 μm, 7.8 × 300 mm, Waters, Milford, MA) driven by a 1200 HPLC pump (Agilent, Santa Clara, CA) and controlled by Eclipse 2 software (Wyatt Technology, Santa Barbara, CA) connected in series to a DAWN Heleos (Wyatt Technology) light scattering photometer and an OptiLab Rex (Wyatt Technology) differential refractive index detector. The mobile phase consisted of 20 mM phosphate buffer (pH 8.0), 100 ppm NaN3, and 0.2 M NaCl (Fisher Scientific) in HPLC grade water (the mean conductivity value being 18.2 × 106 Ω·cm), which was filtered through a 0.2-μm membrane filter. PolybHb solutions were diluted to 1 mg/ml with the mobile phase, and 60 μl of the sample were injected into the column via a 1200 Autosampler (Agilent). All data were collected and analyzed using Astra 5.3 (Wyatt Technology) software.
The O2 affinity and cooperativity coefficient of bHb/PolybHb solutions were regressed from O2-PolybHb equilibrium curves measured on a Hemox Analyzer (TCS Instruments, Southampton, PA) at 37°C.
Samples were prepared by thoroughly mixing 100 μl of the sample with 5 ml Hemox buffer (pH 7.4, TCS Instruments), 20 μl Additive-A, 10 μl Additive-B, and 10 μl antifoaming agent. The PolybHb sample was allowed to equilibrate to a Po2 of 145 ± 2 mmHg using compressed air. After the sample had been given sufficient time to equilibrate, the gas stream was switched to pure N2 to deoxygenate the bHb/PolybHb sample. The absorbance of oxy- and deoxy-Hb (Abs) in solution was recorded as a function of Po2 via dual-wavelength spectroscopy. O2-PolybHb equilibrium curves (Y) were fit to a four-parameter (A0, A∞, P50, n) Hill model (Eq. 1) as follows: (1) where A0 and A∞ are the absorbance at 0 mmHg and full saturation, respectively; Abs is the absorbance of the sample; n is the cooperativity coefficient; and P50 is the Po2 at which the bHb/PolybHb is half-saturated with O2 (O2 affinity).
PolybHb viscosity and COP.
PolybHb viscosity was measured in a cone and plate viscometer DV-II plus with a cone spindle CPE-40 (Brookfield Engineering Laboratories, Middleboro, MA) at a shear rate of 160 s−1. The COP of PolybHb was measured using a Wescor 4420 Colloid Osmometer (Wescor, Logan, UT) (40).
In vivo experiments were performed in 55- to 65-g male Golden Syrian hamsters (Charles River Laboratories, Boston, MA) fitted with a dorsal skinfold window chamber. The hamster window chamber model is widely used for microvascular studies in the unanesthetized state. The complete surgical technique has been described in detail elsewhere (9, 13). Arterial and venous catheters filled with a heparinized saline solution (30 IU/ml) were implanted into the carotid and jugular vessels. Catheters were tunneled under the skin, exteriorized at the dorsal side of the neck, and securely attached to the window frame. Animal handling and care followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the local animal care committee.
The microvasculature was examined 3–4 days after the window implantation surgery, and only animals that passed established systemic and microcirculatory inclusion criteria were used. Animals were considered suitable for experiments if 1) systemic parameters were within normal range, namely, heart rate (HR) > 340 beats/min, mean arterial blood pressure (MAP) > 80 mmHg, systemic Hct > 45%, and arterial Po2 > 50 mmHg; and 2) microscopic examination of the tissue in the chamber observed under ×650 magnification did not reveal signs of low perfusion, inflammation, edema, or bleeding.
The unanesthetized animal was placed in a restraining tube with a longitudinal slit from which the window chamber protruded and then fixed to the microscopic stage for transillumination with the intravital microscope (BX51WI, Olympus, New Hyde Park, NY). Animals were given 20 min to adjust to the tube environment before any measurements were made. The tissue image was projected onto a charge-coupled device camera (4815, COHU, San Diego, CA) connected to a videocassette recorder and viewed on a monitor. Measurements were carried out using a ×40 (LUMPFL-WIR, numerical aperture: 0.8, Olympus) water-immersion objective.
PolybHb solution concentrations were adjusted to 10 g Hb/dl. PolybHb solutions were subdivided by the quaternary state of the bHb during the polymerization reaction, namely, T-state PolybHb and R-state PolybHb. The biophysical properties of both PolybHb solutions are shown in Table 1.
Acute isovolemic exchange transfusion (hemodilution) protocol.
Acute anemia was induced by two isovolemic hemodilution steps. This protocol has been described in detail in our previous reports (4, 37, 38) (Fig. 1). Briefly, the volume of each exchange-transfusion step 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 to 18% by two steps of progressive isovolemic hemodilution using 6% dextran (70 kDa, Pharmacia, Uppsala, Sweden). Blood was simultaneously withdrawn at the same rate from the carotid artery catheter according to a previously established protocol (4, 37). The first exchange was 40% of the blood volume, and the second exchange was 35% of the blood volume, respectively. Moderate hemodiluted animals were randomly divided into two experimental groups (2). The exchange-transfusion protocol was continued by exchanging 35% of the blood volume with the test solution (experimental groups are named accordingly). The duration of the experiments was 4 h. Each exchange and the respective observation time points after the exchange were completed in 1 h. Systemic and microcirculation data were taken after a stabilization period of 15 min.
The two experimental groups were labeled as follows: R-state PolybHb and T-state PolybHb.
Blood chemistry and biophysical properties.
Arterial blood was collected in heparinized glass capillaries (0.05 ml) and immediately analyzed for arterial Po2, arterial Pco2, base excess, and pH (Blood Chemistry Analyzer 248, Bayer, Norwood, MA). The comparatively low arterial Po2 and high arterial Pco2 of these animals are a consequence of their adaptation to a fossorial environment. Blood samples for viscosity and COP measurements were quickly withdrawn from the animal with a heparinized 5-ml syringe at the end of the experiment for immediate analysis. Viscosity was measured in a cone/plate viscometer DV-II plus with a cone spindle CPE-40 (Brookfield Engineering Laboratories) at a shear rate of 160 s−1. COP was measured using a Wescor 4420 Colloid Osmometer (Wescor) (40).
Functional capillary density.
Functional capillary density [FCD; number of capillaries per unit of area (in cm−1)] is the total number of RBC-perfused capillaries divided by the area of the microscopic field of view (37). Capillary segments were considered functional if RBCs were observed to transit over a 60-s period. FCD was tabulated from the number of capillaries with RBC flow in an area composed of 10 successive microscopic fields (420 × 320 μm). Detailed mappings were made of the chamber vasculature to study the same microvessels throughout the experiment.
Arteriolar and venular blood flow velocity were measured online using the photodiode cross-correlation technique (Fiber Optic Photo Diode and Velocity Tracker model 102B, Vista Electronics, San Diego, CA) (20). The centerline velocity was corrected according to vessel size to obtain the mean RBC velocity (V) (25). The video image shearing technique was used to measure vessel diameter (D) online. Blood flow (Q) was calculated from the measured parameters as follows: Q = πV(D/2)2.
Microvascular Po2 distribution.
High-resolution noninvasive microvascular Po2 measurements were made using phosphorescence quenching microscopy (PQM) (21, 37). PQM is based on the O2-dependent quenching of phosphorescence emitted by an albumin-bound metalloporphyrin complex after pulsed light excitation. PQM is independent of the dye concentration within the tissue and is well suited for detecting hypoxia because its decay time is inversely proportional to the Po2 level, causing the method to be more precise at low Po2 levels. This technique is used to measure both intravascular and extravascular Po2 since the albumin-dye complex continuously extravasates from the circulation into the interstitial tissue (21, 37). Tissue Po2 was measured in tissue regions in between functional capillaries. PQM allows for the precise localization of Po2 measurements without subjecting the tissue to injury. These measurements provide a detailed understanding of microvascular O2 distribution and indicate whether O2 is delivered to the interstitial areas.
O2 delivery and extraction.
The microvascular methodology used in our study allows the detailed analysis of O2 delivery to the tissue. Calculations were made using the following equations for O2 delivery (Do2; Eq. 2) and O2 extraction (Vo2; Eq. 3) (4): (2) (3) where RBCHb is the Hb concentration derived from RBCs and is equal to the total Hb − plasma Hb (g Hb/dl blood), PlasmaHb is the concentration of acellular Hb (g Hb/dl blood), γ is the O2 carrying capacity of saturated Hb (1.34 ml O2/g Hb), SA (in %) is the arteriolar RBC O2 saturation, and ŠA (in %) is the arteriolar PolybHb O2 saturation. The subscript A-V indicates arteriolar/venular differences, and Q is the microvascular flow. Fresh hamster RBCs at pH 7.4 and 37.6°C had a P50 (50% of the Hb is saturated with O2) of 32 mmHg and Hill number of 2.9 measured using the Hemox Analyzer (TCS).
Tabular results are presented as means ± SD. The box-whisker plot separates the data into quartiles, with the top of the box defining the 75th percentile, the line within the box giving the median, and the bottom of the box showing the 25th percentile; the upper whisker defines the 95th percentile, and the lower whisker defines the 5th percentile. Data within each group were analyzed using ANOVA for repeated measurements (Kruskal-Wallis test). When appropriate, post hoc analyses were performed with the Dunns multiple-comparison test. Microhemodynamic data are presented as absolute values and ratios relative to baseline values. A ratio of 1.0 signifies no change from baseline, whereas lower and higher ratios are indicative of changes proportionally lower and higher than baseline (i.e., 1.5 would mean a 50% increase from the baseline level). The same vessels and capillary fields were followed so that direct comparisons to their baseline levels could be performed, allowing for more robust statistics for small sample populations. All statistics were calculated using GraphPad Prism 4.01 (GraphPad Software, San Diego, CA). Changes were considered statistically significant if P < 0.05.
Po2 of PolybHb solutions.
bHb was polymerized with glutaraldehyde in the T-state (50:1) and R-state (40:1). Subsequently, each PolybHb mixture was separated into two distinct MW fractions: <500 kDa and >500 kDa. The fraction above 500 kDa in MW was used in this study. For T-state PolybHb, the Po2 was kept at 0 mmHg before and after polymerization and after quenching, which indicates that the bHb was maintained in the fully deoxygenated state (T-state) during the polymerization process. The Po2 of the R-state PolybHb solution before and after polymerization was above the measurement range of the O2 detector. This shows that the bHb was fully saturated with O2 (R-state). After the R-state bHb polymerization reaction was quenched with NaCNBH3 and NaBH4, the Po2 of the R-state PolybHb solution dropped to 0 mmHg, since the NaBH4 generated H2 gas, which displaced all O2 from the solution.
MW distribution of PolybHb solutions.
The results shown in Table 1 demonstrate that the weight-averaged MW of T-state PolybHb was slightly smaller in magnitude compared with R-state PolybHb. Both PolybHb solutions possessed large MWs, ranging from 16.59 to 26.33 MDa, and did not show the presence of bHb in solution.
P50 and n of PolybHb solutions.
The results shown in Table 1 demonstrate that R-state PolybHb had a lower P50 compared with bHb, which itself had a lower value compared with T-state PolybHb. The n values of T-state and R-state PolybHb solutions were <1.
MetHb levels of PolybHb solutions.
The metHb levels of native bHb, T-state PolybHb, and R-state PolybHb are shown in Table 1. The metHb level of native bHb was very low (<1%), since it was purified from fresh bRBCs. T-state PolybHb and R-state PolybHb had similar metHb levels, which were below 5%.
Exchange transfusion with T-state and R-state PolybHb.
Twelve animals were entered into the study; all animals tolerated the entire protocol without visible signs of discomfort. Six animals were assigned to each experimental group. Groups were statistically similar (P > 0.30) in systemic and microcirculation parameters at baseline and moderate hemodilution. Systemic and microhemodynamic datasets for baseline and moderate hemodilution were obtained by combining data from all experimental groups.
Blood chemistry parameters at baseline, moderate hemodilution, and after the exchange transfusion are shown in Table 2. Moderate hemodilution reduced the Hct and Hb concentrations. The Hct for all experimental groups was similar (11.3 ± 0.4%). Total Hb was increased from the moderate hemodilution level after exchange with T-state and R-state PolybHb. The Hct after the exchange transfusion remained stable over time until the end of the experiment for both experimental groups.
Results from the systemic arterial blood gas analysis are shown in Table 2. The arterial Po2 was statistically significantly increased from baseline after moderate hemodilution. The exchange transfusion with T-state and R-state PolybHb statistically significantly increased the arterial Po2 compared with baseline and moderate hemodilution, respectively. However, the arterial Po2 after the exchange transfusion with T-state PolybHb was statistically lower than after the exchange transfusion with R-state PolybHb. The arterial Pco2 decreased significantly from baseline after moderate hemodilution. The exchange transfusion with T-state and R-state PolybHb further increased the arterial Pco2 compared with baseline and moderate hemodilution. The exchange with R-state PolybHb had a more pronounced increase in arterial Pco2 compared with T-state PolybHb. Arterial pH was higher than baseline after moderate hemodilution, and the exchange transfusion with T-state and R-state PolybHb maintained a higher arterial pH compared with baseline. The blood acid-base balance was statistically significantly decreased after moderate hemodilution and in both experimental groups compared with baseline. The exchange transfusion with R-state PolybHb produced severe blood acid-base unbalance compared with T-state PolybHb.
Changes in MAP and HR are shown in Fig. 2. MAP was statistically decreased from baseline after moderate hemodilution. The exchange transfusion with T-state PolybHb maintained the MAP achieved after moderate hemodilution and was statistically lower than baseline, whereas R-state PolybHb increased MAP compared with moderate hemodilution and remained statistically lower than baseline. HR was no different from baseline after moderate hemodilution, and it remained no different after the exchange transfusion with PolybHb solutions.
Blood biophysical properties after exchange.
Blood viscosity, plasma viscosity, and plasma COP after hemodilution for baseline and both experimental groups are shown in Table 3. Blood viscosities were lower than baseline for both exchange-transfused groups. Animals exchange transfused with T-state PolybHb had statistically higher blood and plasma viscosity than animals exchange transfused with R-state PolybHb. Plasma viscosity did not change from baseline. Plasma COP was lower for both exchange-transfused groups compared with baseline.
Microvascular parameters were characterized for large feeding and small arcading arterioles (range: 45–74 μm) and small collecting venules (range: 46–76 μm). Changes in diameter and blood flow from baseline, as well as absolute values, are shown Fig. 3. Arterioles after moderate hemodilution were dilated, and RBC velocity and blood flow were statistically increased from baseline. Arterioles were statistically constricted for both exchange-transfused groups compared with moderate hemodilution but no different from baseline. Arteriolar blood flows after moderate hemodilution were statistically higher than baseline. Arteriolar blood flows after exchange transfusion with T-state and R-state PolybHb were statistically lower compared with moderate hemodilution but no different from baseline. Animals exchange transfused with R-state PolybHb had statistically lower arteriolar blood flows than animals exchange transfused with T-state PolybHb.
Venular microvascular tone and blood flow changes are also shown in Fig. 3. Moderate hemodilution did not produce dilation; however, it statistically increased RBC velocity and blood flow compared with baseline. Venular blood flows after the exchange transfusion decreased compared with moderate hemodilution in both groups but were no different from baseline. Shear rate and shear stress values for all experimental groups are shown in Table 3.
FCD values after moderated hemodilution and the exchange transfusion are shown in Fig. 4. Moderated hemodilution decreased FCD compared with baseline. The exchange transfusion with T-state and R-state PolybHb further decreased FCD compared with baseline and moderate hemodilution. The exchange transfusion with R-state PolybHb produced lower FCD than the exchange transfusion with T-state PolybHb.
Microvascular O2 tensions.
Microvascular and tissue Po2 values are shown in Fig. 5. The exchange transfusion with R-state PolybHb produced lower arterial O2 tensions than the exchange transfusion with T-state PolybHb. Similar results were found for interstitial tissue O2 tensions and venular O2 tensions. Interstitial tissue Po2 in both experimental groups was lower compared with the normal tissue Po2 without hemodilution (full Hct), which is 21.7 ± 3.5 mmHg (8).
Microvascular O2 delivery and extraction.
Figure 6 shows resulsts from the analysis of systemic and microvascular O2 delivery and extraction. Systemic O2 delivery was 8% higher for T-state PolybHb than for R-state PolybHb. Premicrocirculation O2 extraction was statistically higher for T-state PolybHb compared with R-state PolybHb. Arteriolar O2 delivery after extreme hemodilution with T-state or R-state PolybHb was no different. Microcirculation O2 extraction was statistically higher for T-state PolybHb compared with R-state PolybHb. The venular O2 reserve was statistically higher for R-state PolybHb compared with T-state PolybHb.
Systemic O2 extraction from the remaining RBCs after hemodilution was statistically higher after the exchange with R-state PolybHb compared with T-state PolybHb. Animals exchanged with R-state PolybHb extracted 98% of O2 in the RBCs compared with 80% for the animals exchanged with T-state PolybHb. Systemic O2 extraction from the polymerized Hb in the plasma was 70% for animals exchanged with T-state PolybHb compared with 16% for animals exchanged with R-state PolybHb. A similar result was observed at the microvascular level, where animals exchanged with T-state PolybHb extracted 75% of O2 available in RBCs and 59% of O2 available in T-state PolybHb compared with R-state PolybHb, in which 97% of O2 available in RBCs and 12% of O2 available in R-state PolybHb was extracted.
Biophysical properties of T-state and R-state PolybHb solutions.
The goal of this study was to examine the role of high-MW PolybHb O2 affinity (P50) in regulating tissue oxygenation. We hypothesized that the P50 of a PolybHb solution is a major determinant of tissue oxygenation. To test this hypothesis, two types of high-MW PolybHb solutions were synthesized with different initial quaternary states (T and R states) and used for the in vivo evaluation of systemic and microcirculatory parameters.
bHb was polymerized under anaerobic and aerobic conditions. For T-state PolybHb, the polymerization process minimized the oxidation of the heme since bHb was polymerized in an anoxic environment (Po2 = 0 mmHg) and quenched with the strong reducing agent NaBH4. For R-state PolybHb, bHb was polymerized in a saturated O2 environment (Po2 > 749 mmHg), and some of the oxidized bHb was able to be reduced after quenching with both the mild reducing agent NaCNBH3 and the strong reducing agent NaBH4. As a further precaution against the autooxidation of the heme, PolybHb solutions were buffer exchanged against modified lactated Ringer solution containing N-acetyl-l-cysteine, an antioxidant (43), which limits heme oxidation. T-state and R-state PolybHb solutions possessed very similar weight-averaged MWs (Table 1), although R-state PolybHb had a slightly higher weight-averaged MW versus T-state PolybHb. This can be easily explained by the presence of Na2S2O4 in the T-state bHb polymerization reaction. During the T-state bHb polymerization process, extra Na2S2O4 was titrated into the bHb solution after the Po2 attained a value of 0 mmHg to maintain the bHb solution in the T state during the glutaraldehyde polymerization process. The excess Na2S2O4 quenched some of the glutaraldehyde in solution, thus reducing the weight-averaged MW of the T-state PolybHb solution compared with the R-state PolybHb solution.
For T-state PolybHb, the Po2 of the initial bHb solution was maintained at 0 mmHg before polymerization. Therefore, all bHb molecules in solution were maintained in the low-O2 affinity T state. After polymerization of T-state bHb, the reducing agent NaBH4 was used to quench any remaining glutaraldehyde in solution as well as to reduce the resulting Schiff bases. Hence, T-state PolybHb was frozen in the low-O2 affinity state via intra- and intermolecular glutaraldehyde cross-links within the bHb tetramer and between tetramers. The P50 of high-MW T-state PolybHb (40 mmHg) was statistically different to that of unmodified bHb (26 mmHg) (30) and similar to the reported value of 38 mmHg for Hemopure (a glutaraldehyde cross-linked bHb manufactured by Biopure) (36). In the case of R-state PolybHb, bHb was maintained in the R state (high O2 affinity) during the polymerization process and subsequently quenched with both NaCNBH3 and NaBH4. Therefore, R-state PolybHb was frozen in the high-O2 affinity R state via intra- and intermolecular glutaraldehyde cross-links within the bHb tetramer and between tetramers. The P50 of R-state PolybHb is 1 mmHg. The cooperativity of the two PolybHb solutions is much less compared with the reported value for unmodified bHb (2.5) (30). This behavior is explained by the glutaraldehyde cross-links present within and between the globin chains, which restrict the transmission of any quaternary changes in the Hb structure to other neighboring globin chains within the Hb tetramer. This results in a significant loss of cooperative binding of O2 molecules to the Hb tetramer.
Exchange transfusion with T-state and R-state PolybHb.
The principal finding of this study was that under identical extreme anemic conditions, bHb polymerized in the T state (deoxygenated; T-state PolybHb: P50 = 40 mmHg) significantly improved systemic and microvascular oxygenation compared with bHb polymerized in the R state (oxygenated; R-state PolybHb: P50 = 1 mmHg). The appreciably increased tissue Po2 attained with T-state PolybHb appears to be due to 1) the higher amount of O2 released from the PolybHb; 2) the maintenance of macrovascular hemodynamics (e.g., blood pressure and HR); and 3) the microvascular hemodynamics (e.g., vascular tone and blood flow) compared with R-state PolybHb.
In the initial phase of the protocol to reach the moderate hemodilution state, blood was exchanged with dextran solution as a colloidal plasma expander to lower the intrinsic O2 carrying capacity (Hct) of blood, which also changes the rheological properties of blood. The compensatory mechanisms responding to the acute decrease in Hct involve the increase of cardiac output due to a reduction in vascular resistance due to a lower blood viscosity (10, 14, 18, 23). During hemodilution, blood viscosity is an important factor in the responses of the cardiovascular system, as it affects endothelial shear stress, which activates the synthesis of vascular autocoids, such as prostacyclin and NO (22, 26, 27, 35, 42). Beyond the moderate hemodilution level, exchange transfusion using PolybHb at 10 g/dl increases blood O2 content and plasma viscosity; however, blood viscosity remains lower than nonhemodiluted blood. Mechanistically, from a rheological standpoint, further decreases in Hct paired with an increase in plasma viscosity with high-viscosity O2 carriers can partially preserve vascular shear stress stimuli plus sustain oxygenation. Thus, T-state PolybHb acted as a viscogenic O2 carrier, maintaining both shear stress and oxygenation, without increasing vascular resistance consequent to an increase in plasma viscosity, leading to an improved transmission of central pressure to the microcirculation sustaining FCD.
Exchange transfusion with either T-state or R-state PolybHb resulted in a higher total Hb in the circulation (6.2 and 6.1 g/dl) than moderate hemodilution (5.8 g/dl). However, arterial blood gases (Po2 and Pco2) were drastically changed after the exchange transfusion, which suggests significant adjustments in the way that the respiratory system exchanged gases as a result of the presence of PolybHb in the blood. In an attempt to improve oxygenation, animals exchange transfused with R-state PolybHb increased arterial Po2, which only increased the dissolved O2 within the plasma without affecting O2 transported or delivered by the remaining RBCs and R-state PolybHb. Moreover, the calculated O2 extraction for animals exchanged transfused with R-state PolybHb showed that almost all the O2 loaded into the RBCs was extracted, and only a small fraction of the O2 in R-state PolybHb was ever released to hypoxic tissues. These results suggest that extremely high O2 affinity of R-state PolybHb prevents the O2 bound to R-state PolybHb from been used by the tissues. Since both groups had the same Hct after the exchange transfusion, changes in O2 delivery and extraction are mostly due to the biophysical properties of the PolybHb. It appears that O2 affinity appears to be a limiting factor for the effective transport and release of O2.
The differences in oxygenation, tissue Po2, MAP, and arteriolar blood flow between T-state and R-state PolybHb shows that in designing a HBOC, it is not sufficient to provide adequate O2 carrying capacity. Once a suitable O2 carrier is available, it must also be able to maintain or enhance other circulatory transport parameters, particularly blood flow. The large polymers of bHb used in this study are practically vasoinactive, and the difference in P50 appears to be the determining factor involved in oxygenation. Introducing into the circulation PolybHb with P50 slightly higher than the hamster blood (P50 = 32 mmHg) will deliver O2 earlier to the tissues and preserve O2 in the RBCs to be released to the tissues that need it. On the other hand, the presence of even considerable amounts of strongly left-shifted Hbs would prevent O2 transport in the organism.
In summary, the results presented here show that T-state PolybHb is an efficient high-viscosity O2 carrier. When used during extreme anemic conditions, it produced a superior systemic and microvascular hemodynamic and oxygenation outcome compared with R-state PolybHb. The effects on microvascular hemodynamics were comparable, if not better, than those previously reported in a similar protocol (Table 4). These results show that maintaining blood rheological properties during extreme anemic conditions by means of the high-viscosity PolybHb increases oxygenation beyond the already observed benefit with a high-viscosity plasma expander (6% 500-kDa dextran). Previous results using high-viscosity plasma expanders include vasodilation and increased microvascular flow (37), sustenance of capillary pressure, maintenance of FCD (7), increased production of endothelium-mediated factors (35), and enhanced organ flow (6), effects that we speculate remain partially valid for T-state PolybHb. The potentially negative effect of increasing peripheral vascular resistance due to the increase in plasma and blood viscosity was compensated for by the already anemic state of the animals and improvement of microvascular function.
This work was supported by Bioengineering Research Partnership (National Heart, Lung, and Blood Institute) Grant R24-HL-64395, Program Project P01-HL-071064, and Grants R01-HL-078840, R01-HL-62354, R01-HL-62318, R01-HL-76182, R01-HL-078840, and R01-DK070862.
No conflicts of interest are declared by the author(s).
The authors greatly acknowledge A. Barra and C. Walter (University of California-San Diego) for technical assistance.
- Copyright © 2010 the American Physiological Society