AJP - Heart BIOPAC complete lab solutions
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 295: H2212-H2218, 2008. First published October 3, 2008; doi:10.1152/ajpheart.00751.2008
0363-6135/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/5/H2212    most recent
00751.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cabrales, P.
Right arrow Articles by Intaglietta, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cabrales, P.
Right arrow Articles by Intaglietta, M.

Isovolemic exchange transfusion with increasing concentrations of low oxygen affinity hemoglobin solution limits oxygen delivery due to vasoconstriction

Pedro Cabrales,1 Amy G. Tsai,1,2 and Marcos Intaglietta2

1La Jolla Bioengineering Institute and 2Department of Bioengineering, University of California, San Diego, La Jolla, California

Submitted 18 July 2008 ; accepted in final form 29 September 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
O2-carrying fluids based on hemoglobin (Hb) are in various stages of clinical trials to determine their suitability as O2-carrying plasma expanders. Polymerized Hb solutions are characterized by their vasoactivity, low O2 affinity, oncotic effect, prolonged shelf life, and stability. Physiological responses to facilitated O2 transport after exchange transfusion with polymerized bovine Hb (PBH) were studied in the hamster window chamber model during acute moderate anemia to determine how PBH affects microvascular perfusion and tissue oxygenation. The anemic state [29% hematocrit (Hct)] was induced by hemodilution with a plasma expander (70 kDa dextran). After hemodilution, animals were randomly assigned to different exchange transfusion groups. Study groups were based on the concentration of PBH used, namely: PBH at 13 g Hb/dl [PBH13], PBH diluted to 8 (PBH8) or 4 (PBH4) g Hb/dl in albumin solution at matching colloidal osmotic pressure (COP), and no PBH (only albumin solution) at matching COP (PBH0). Measurement of systemic parameters, microvascular hemodynamics, capillary perfusion, and intravascular and tissue O2 levels was performed at 18% Hct. Restitution of O2-carrying capacity with PBH13 increased arterial pressure and triggered vasoconstriction, low perfusion, and high peripheral resistance. PBH4 and PBH0 exhibited lower arterial pressures compared with PBH13. Exchange transfused animals with PBH8 and PBH4 better maintained perfusion and functional capillary density than PBH13. Blood gas parameters and acid-base balance were recovered proportional to microvascular perfusion. Arterial O2 tensions were improved with PBH4 and PBH8 by preventing O2 precapillary release and increasing O2 reserve. Further studies to establish PBH optimal dosage, efficacy, safety, and its effect on outcome are indicated before Hb-based O2-carrying blood substitutes are implemented in routine practice.

microcirculation; red blood cells; hemodilution; functional capillary density; hemoglobin oxygen affinity; tissue oxygen

BLOOD SUBSTITUTES ARE in continued development, driven in part by the public perception that the blood supply is not safe and looming shortages (18). However, blood safety, related to the transmission of viral diseases, is no longer a major concern in the United States, and blood shortages have not materialized due to continued development of surgical techniques that reduce blood losses and lower transfusion triggers (18). Despite these advances, the development of effective blood substitutes that maintain blood volume (BV) and deliver O2 remains a priority for emergency combat care, as well as for severe blood losses associated with trauma. Blood is a vital public health resource that must be readily available at all times, especially in the event of a disaster.

Hemoglobin (Hb)-based O2-carrying blood substitutes (HBOCs) are available in an impressive range of structural modifications and formulations arising from trying to engineer into the products varied physiological characteristics deemed important for the targeted clinical application. The final products are formulated as solutions with Hb concentration ranging from 4 to 14 g/dl, with limitations due to colloidal osmotic pressure (COP), viscosity, and production efficiency, leading to products with unique O2 delivery characteristics and physiological responses.

Initial experimental results with intravenous infusion of acellular unmodified Hb in animals and humans were reviewed by Sellards and Minot (23). The early studies showed toxicity due to stroma [debris of red blood cell (RBC) membranes], rapid oxidation of Hb, rapid loss of Hb from the blood stream into the urine (resulting from kidney damage), and an elevation of blood pressure and reduction in heart rate (HR, even with very low doses). Current HBOC eliminated these toxicities by extensive purification and protein stabilization. However, products remain vasoactive, an effect attributed to nitric oxide scavenging, extravasation, and vasculature hyperoxygenation (29). Hypertension is the most frequent clinical manifestation of vasoconstriction and appears to be the main unwanted side effect of HBOCs. On the other hand, they have O2-carrying capacity similar to blood and can also increase O2 transport to tissues by facilitated O2 diffusion. Our research, focused on the analysis of effects at the microvascular level, has demonstrated that restitution of O2-carrying capacity must be performed in conjunction maintenance of perfusion to maintain/increase O2 delivery (5).

At anemic states near the critical hematocrit (Hct), the organism maintains O2 delivery via the remaining circulating O2-carrying capacity and the enhanced perfusion, due to reduction of blood viscosity and increased cardiac output (2). At this point, maintenance of adequate oxygenation is particularly sensitive to further reductions in Hct, and to systemic or local changes in O2 demand, such as the extra myocardial work needed to maintain peripheral O2 delivery. The objective of this study was to identify the concentration effects of low O2 affinity Hb on blood perfusion and oxygenation at moderate anemic conditions. To achieve this objective, the hamster chamber window model was subjected to moderate hemodilution to Hct of 28% using a plasma expander (6% dextran 70 kDa; Pharmacia, Uppsala, Sweden). Hemodilution was continued to 18% Hct using different concentrations of polymerized bovine Hb (PBH; Oxyglobin; Biopure, Cambridge, MA; approved in the United States and European Union for veterinary use). PBH concentrations tested were 0, 4, 8 and 13 g Hb/dl at matching COP. COP was adjusted using human serum albumin diluted in normotonic saline.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal preparation. Investigations 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 is described in detail elsewhere (9, 10). Arterial and venous catheters filled with a heparinized saline solution (30 IU/ml) were implanted in 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.

Inclusion criteria. The microvasculature was examined 3–4 days after the window implantation surgery, and only animals passing an established systemic and microcirculatory inclusion criteria we used. Animals were suitable for the experiments if: 1) systemic parameters were within normal range, namely, HR >340 beat/min, mean arterial blood pressure (MAP) >80 mmHg, systemic Hct >45%, and arterial O2 partial pressure (PaO2) >50 mmHg; and 2) microscopic examination of the tissue in the chamber observed under x650 magnification did not reveal signs of low perfusion, inflammation, edema, or bleeding. Hamsters are a fossorial species with a lower PaO2; however, their microvascular PO2 distribution in the chamber window model is similar to other rodents (4).

Test solutions. PBH was commercially available as Oxyglobin (Biopure). PBH is produced by nonspecific polymerization with glutaraldehyde (mean mol mass 200 kDa) in a modified lactated Ringer solution. PBH (Oxyglobin) has a concentration of 13 g Hb/dl (COP: 38 mmHg, viscosity: 1.9 cP). Other PBH concentrations tested were 0, 4, and 8 g Hb/dl. COP of all solution was balanced using human serum albumin diluted in normal saline (~38 mmHg). PBH solution viscosities for 0, 4, and 8 g Hb/dl were 1.2, 1.3, and 1.6 cP, respectively.

Acute isovolemic exchange transfusion (hemodilution) protocol. Acute anemia was induced by one isovolemic hemodilution step. Briefly, the volume of each exchange-transfusion step was calculated as a percentage of BV, estimated as 7% of body weight. Mild hemodilution (MH) induced by isovolemic exchange was 40% of BV, using 6% dextran 70 kDa (Pharmacia). After induction of MH, animals were randomly divided into four experimental groups (1). The exchange transfusion protocol was continued by exchanging 35% of the BV with the text solution (Fig. 1).


Figure 1
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 1. Hemodilution was attained by means of a progressive, stepwise, isovolemic hemodilution protocol in which the red blood cell (RBC) volume (filled bar) is continuously decreased and the plasma volume is increased (open bar) while maintaining the total blood volume constant (represented by the dotted line). The volume of each exchange-transfusion step was calculated as a percentage of the blood volume, estimated as 7% of the body weight. A mild moderate anemic state was induced by lowering systemic hematocrit (Hct) by one progressive isovolemic hemodilution (40% of the blood volume) using 6% dextran 70 [viscosity of 2.8 cP and colloidal oncotic pressure (COP) of 50 mmHg], labeled mild hemodilution (MH). The final anemic state was achieved by a second hemodilution step (35% of blood volume) using either 0, 4, 8, or 13 g/dl of polymerized bovine hemoglobin (PBH). COP of all solutions was balanced using human serum albumin diluted in normal saline (38 mmHg). PBH solution viscosities for 0, 4, 8, and 13 g/dl were 1.2, 1.3, 1.6, and 1.9 cP, respectively.

 
Blood was simultaneously withdrawn at the same rate from the carotid artery catheter according to a previously established protocol (3, 7, 27). Blood samples were withdrawn at the end of the experiment for subsequent analysis of viscosity and COP. Each exchange and the respective observation time point postexchange were completed in 1 h. Systemic and microcirculation data were taken after a stabilization period of 15 min.

Experimental groups. The experimental groups were labeled PBH0, PBH4, PBH8, and PBH13 based in the concentration of PBH used: group 1, PBH13, exchange transfusion with stock solution of PBH (Oxyglobin); group 2, PBH8, exchange transfusion with PBH diluted to 8 g Hb/dl; group 3, PBH4, exchange transfusion with PBH diluted to 4 g Hb/dl; and group 4, PBH0, exchange transfusion without PBH.

Systemic parameters. MAP and HR were recorded continuously (MP 150; Biopac System, Santa Barbara, CA). Hct was measured from centrifuged arterial blood samples taken in heparinized capillary tubes. Hb content was determined spectrophotometrically (B-Hemoglobin; Hemocue, Stockholm, Sweden).

Blood chemistry and biophysical properties. Arterial blood was collected in heparinized glass capillaries (50 µl) and immediately analyzed for PO2, PCO2, base excess (BE), and pH (Blood Chemistry Analyzer 248; Bayer, Norwood, MA). Viscosity was measured in a DV-II plus (Brookfield, Middleboro, MA). Plasma COP was measured using a 4420 Colloid Osmometer (Wescor, Logan, UT).

Microvascular experimental setup. 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 measurement. The tissue image was projected on a charge-coupled device camera (COHU 4815) connected to a videocassette recorder and viewed on a monitor. Measurements were carried out using a x40 (LUMPFL-WIR, numerical aperture 0.8; Olympus) water immersion objective.

Functional capillary density. Functional capillaries, defined as those capillary segments that have RBC transit of at least a single RBC in a 45-s period in 10 successive microscopic fields, were assessed, totaling a region of 0.46 mm2. The relative change in functional capillary density (FCD) from baseline levels after each intervention is indicative of the extent of capillary perfusion (3).

Microhemodynamics. Arteriolar and venular blood flow velocities were measured on-line by using the photodiode cross-correlation method (Photo Diode/Velocity Tracker Model 102B; Vista Electronics, San Diego, CA). The measured centerline velocity (V) was corrected according to vessel size to obtain the mean RBC velocity. A video image-shearing method was used to measure vessel diameter (D) (14). Blood flow (Q) was calculated from the measured values as Q = {pi} x V (D/2)2. This calculation assumes a parabolic velocity profile and has been found to be applicable to tubes of 15–80 µm internal diameters and for Hcts in the range of 6–60% (17).

Microvascular PO2 distribution. High-resolution noninvasive microvascular PO2 measurements were made using phosphorescence quenching microscopy (PQM) (16, 27). PQM is based on the O2-dependent quenching of phosphorescence emitted by 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 values. 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 (16, 27). Tissue PO2 was measured in tissue regions in between functional capillaries. PQM allows for precise localization of the 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.

Oxygen delivery and extraction. The microvascular methodology used in our studies allows a detailed analysis of O2 delivery to the tissue. Calculations are made using Eq. 1 for O2 delivery (DO2) and Eq. 2 for O2 extraction (VO2) (3).

Formula 1(1)

Formula 2(2)
where RBCHb is the Hb from RBCs equal to total Hb – plasma Hb (g Hb/dl blood), PlasmaHb is the acellular Hb (g Hb/dl blood), {gamma} is the O2-carrying capacity of saturated Hb (1.34 ml O2/g Hb), SA is the arteriolar RBC O2 saturation, SA is the arteriolar PBH O2 saturation, SA-V indicates the 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 a Hemox Analyzer (TCS). PBH is only 72% saturated at 150 mmHg; therefore, P50 is 54 mmHg with a Hill number of 1.2 (6).

Data analysis. Tabular results are presented as means ± SD. The box-whisker plot separates the data into quartiles, with 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 top whisker defines the 95th percentile and lower whisker the 5th percentile. Data within each group were analyzed using analysis of variance for repeated measurements (ANOVA, Kruskal-Wallis test). When appropriate, post hoc analyses were performed with the Dunn's 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.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Twenty-four animals were entered into the study; all animals tolerated the entire hemodilution 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 MH. Systemic and microhemodynamic datasets for baseline and MH were obtained by combining data from all experimental groups.

Systemic parameters. Hct, Hb, plasma Hb, MAP, HR, and blood chemistry for the study are presented in Table 1. Hct was not different between experimental groups. Total Hb was increased, proportional to the concentration of PBH used during exchange transfusion. PBH8 and PBH13 had total Hb levels higher than MH, >2 g Hb/dl in plasma. Hct and Hb after exchange transfusion with a different concentration of PBH remained stable over time until the end of the experiment.


View this table:
[in this window]
[in a new window]

  		Table 1. Systemic parameters during the exchange protocol

 
MAP was statistically decreased from baseline after MH. Exchange transfusion affected MAP proportional to the concentration of PBH. All animals that received PBH statistically recovered MAP from MH, with PBH13 showing an increase in pressure from baseline. MAP for PBH4 and PBH8 were not different from MH. PBH0 had lower MAP than all experimental groups with PBH in the plasma (PBH4, PBH8, and PBH13).

Arterial blood gases are presented in Table 1. Arterial PO2 was statistically significantly increased from baseline after MH and in all experimental groups. Arterial PCO2 decreased significantly from baseline for all experimental groups. Arterial PO2, PCO2, and pH were not different among experimental groups. Blood BE was statistically decreased for all experimental groups compared with baseline. All groups with PBH in the plasma maintained positive acid-base balance, and most parameters were statistically improved when compared with PBH0 (P < 0.10).

Blood biophysical properties after exchange. Blood viscosity, plasma viscosity, and plasma COP for all experimental groups are presented in Table 2. Blood viscosities were lower than baseline for all exchange-transfused groups, with no differences among groups. This further supports the importance of matching colloid properties to perform a fair comparison of different concentrations of PBH. Plasma viscosity did not change from baseline. Plasma COP was higher from baseline for all exchange-transfused groups.


View this table:
[in this window]
[in a new window]

  		Table 2. Blood rheological properties, WSR, and WSS

 
Microhemodynamics. Microvascular parameters were characterized for large feeding and small arcading arterioles (range 44–72 µm) and small collecting veins and venules (range 48–74 µm). The changes in diameter, RBC velocity, and blood flow from baseline are presented in Fig. 2. Absolute values are given in the legend for Fig. 2. Arteriole diameter after moderate hemodilution remained unchanged from baseline, 1.07 ± 0.06 of baseline; RBC velocities were 1.19 ± 0.26 of baseline and blood flow 1.36 ± 0.34 of baseline (n = 112 vessels). Arterioles were statistically constricted for PBH13 when compared with baseline and other experimental groups (PBH0, PBH4, and PBH8). Arteriolar RBC velocity was statistically higher than baseline for all groups, and PBH4 had higher RBC velocity than the other groups. Arteriolar blood flows were statistically higher for PBH4 and PBH0 when compared with PBH8 and PBH13.


Figure 2
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 2. Relative changes to baseline in arteriolar and venular hemodynamics after hemodilution, followed by exchange transfusion with different concentrations of PBH. Broken line represents baseline level. {dagger}P < 0.05 and {ddagger}P < 0.05, relative to baseline. Diameters (µm, mean ± SD) in each animal group were as follows: baseline [arterioles (A): 66.1 ± 8.3, n = 52 vessels; venules (V): 65.6 ± 8.8, n = 60]. RBC velocities (mm/s, mean ± SD) in each animal group were as follows: baseline (A: 4.6 ± 0.9; V: 1.8 ± 0.7). Flow (nl/s, mean ± SD) in each animal group was as follows: baseline (A: 14.3 ± 4.7; V: 6.9 ± 2.8).

 
Venular microvascular tone, RBC velocity, and blood flow changes are presented in Fig. 2. Moderate hemodilution produced minor venular hemodynamic alterations, diameters were 1.02 ± 0.09 of baseline, RBC velocities were 1.28 ± 0.23 of baseline, and blood flow was 1.35 ± 0.34 of baseline (n = 118). Exchange transfusion with different concentrations of PBH produced increased venular RBC velocity for all groups. Venular RBC velocities for PBH4 were statistically higher than PBH0. Venular blood flows for PBH0 and PBH4 were higher than baseline and PBH13. Shear rate and shear stress for all experimental groups are presented in Table 2.

FCD. FCD after hemodilution and exchange transfusion with different PBH concentration are presented in Fig. 3. All exchange-transfused groups had lower FCD than baseline. PBH13 had statistical lower FCD than all the other exchange-transfused groups. FCD for PBH0, PBH4, and PBH8 was not different between groups.


Figure 3
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 3. Functional capillary density (FCD) after hemodilution followed by exchange transfusion with different concentrations of PBH. All values are relative to baseline levels. FCD (cm–1) at baseline was 117 ± 18. *P < 0.05.

 
Microvascular oxygen tensions. Microvascular and tissue O2 tensions are shown in Fig. 4. PBH4 and PBH8 yielded high PaO2 when compared with PBH0 and PBH13. Venular PO2 were not different among groups. Exchange with PBH4 produced higher tissue PO2 than other groups. PBH8 presented higher extravascular PO2 than PBH13. PBH0 and PBH13 showed lower tissue PO2 from normal at full Hct, 21.7 ± 3.5 mmHg (8); in the other groups, tissue PO2 was not different from normal at full Hct.


Figure 4
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 4. Intravascular and extravascular partial pressure of O2 after hemodilution followed by exchange transfusion with different concentrations of PBH. *P < 0.05.

 
Microvascular oxygen delivery and extraction. Figure 5 shows analysis of systemic and microvascular O2 delivery and extraction. Total systemic O2 delivery (Fig. 5C) was higher for PBH4 than PBH0, PBH8, and PBH13, and total systemic O2 extraction (Fig. 5F) was similar among all groups. The fraction of total O2 delivery provided by PBH increased proportional to its concentration, as expected. However, it also decreased from RBC as the concentration increased. Premicrocirculation O2 delivery (Fig. 5A) was statistically higher for PBH13 when compared with PBH0, PBH4, and PBH8. Premicrocirculation O2 delivery provided by PBH and RBC increased proportional to the concentration of PBH. Microcirculation O2 delivery (Fig. 5B) was statistically higher for PBH4 when compared with PBH0, PBH8, and PBH13. The fraction of microcirculation O2 delivery provided by PPB increased proportional to the concentration of PBH, and, from RBC, it initially decreased and then increased as concentration increased. Total O2 extraction was not different among all the exchange-transfused groups. Premicrocirculation O2 extraction (Fig. 5D) was higher for PBH0 and PBH13 compared with PBH4 and PBH8. The opposite was found for microcirculatory O2 extraction (Fig. 5E), where PBH4 and PBH8 were higher than PBH0 and PBH13. The systemic RBC-to-O2 extraction ratio (delivery over extraction) was lower for PBH4 compared with the other groups. Plasma Hb extraction ratio was higher after exchange with PBH13 compared with PBH4 and PBH8.


Figure 5
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 5. Premicrocirculation, microcirculation, and total O2 delivery and extraction. A: premicrocirculation O2 delivery. B: microcirculation O2 delivery. C: total O2 delivery. D: premicrocirculation O2 extraction. E: microcirculation O2 extraction. F: total O2 extraction. Black areas correspond to RBC contribution (~6 g/dl), and gray areas correspond to PBH contribution. Total, premicrocirculation, and microcirculation O2 delivery and extraction were calculated from systemic and microcirculatory parameters.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The principal finding of the study is that the optimal concentration of low O2 affinity HBOC in plasma capable of enhancing microvascular O2 transport is determined by how it affects hemodynamic conditions and not by the amount of Hb infused. The presence of low-concentration PBH (~1.2 g Hb/dl) in the circulation was more effective in increasing oxygenation than medium or large concentrations (>2 g Hb/dl), or in the absence of PBH. During exchange transfusion with PBH, maintenance of systemic and microvascular hemodynamics is crucial, because a reduction in perfusion directly decreases oxygenation by canceling any benefit from the PBH and even the remaining RBCs. To positively affect O2 delivery and tissue PO2, an HBOC must be a good plasma expander. Colloidal osmotic effects of different concentrations of PBH were not a factor during the study because solution COP was matched before exchange transfusion and verified by similarities in Hct in all groups. These also eliminate any differences in hamsters O2 transport capacity (Hct) within groups, suggesting that all differences found in these studies are due to changes in plasma O2-carrying capacity from PBH.

Isovolemic hemodilution with non-O2 carrier plasma expander leads to changes in systemic and hemodynamic conditions that compensate for the reduction in Hct (O2-carrying capacity). At Hb concentration of 6.0 g/dl, the organism is nearly the limit to maintain O2 delivery. Interestingly, an increase in Hb concentration from 6.0 to 7.8 g/dl with RBC transfusion is capable of reverting hemodilution-induced dysfunctions (24). Other studies have shown that tolerance to moderate hemodilution can be effectively managed with hyperoxic ventilation, which improves tissue oxygenation and survival (15, 19). These beneficial effects have been attributed to an increase in arterial O2 content via an increase of physically dissolved O2 in the plasma, an increase in MAP, and coronary perfusion pressure, due to hyperoxic arteriolar vasoconstriction. On the other hand, exchange transfusion with different concentrations of PBH increased MAP and vascular resistance in proportion to plasma concentration. However, microvascular flow did not correlate with Hb plasma concentration, where 1.2 g/dl of PBH maintained perfusion and increased tissue O2 delivery. Our results show the importance of an optimal dose of PBH to ensure maintenance of hemodynamic conditions and effectively utilize the added O2-carrying capacity.

The significant increase in microvascular oxygenation observed with 1.2 g/dl of PBH is the result of preserving the O2 in the blood until it arrives to the microcirculation. In the absence of plasma Hb, blood released 25% of the total O2 before arrival to the microcirculation, and the remaining 75% is delivered to the microcirculation, which extracts 65% of this O2 and returns the rest to the circulating O2 pool. This equilibrium was affected by the presence of 1.2 g of PBH per deciliter in plasma by preventing the release of O2 from RBCs before microcirculation, where only 13% of total O2 delivery was released before the arrival of blood to the microcirculation. Because the total amount of O2 extracted was similar for all groups, animals exchanged with PBH4 had the largest O2 reserve (the difference between total O2 delivery and total O2 extracted; Fig. 5, C and F). In other words, 1.2 g/dl in plasma preserved O2 in the blood to be released in larger proportion in the microcirculation, which extracts what it needs and returns the excess. Plasma PBH concentrations >1.2 g/dl favored the release of O2 before the microcirculation, which could trigger an O2 regulatory mechanism causing an increase in vascular tone (vasoconstriction) and hypoperfusion.

Moderated supplementation of plasma O2-carrying capacity PBH solutions prevented physiological autoregulation of arteriolar O2 release (13). It is well established that vascular smooth muscle cells are O2 sensors containing the early responsive elements, which adjust blood flow to tissue O2 demand (12). During moderate anemic conditions, the remaining RBCs sustain O2 supply if perfusion is increased; however, conventional plasma expanders are designed to dilute blood and not to enhance cardiovascular function. In the case of HBOCs, the two factors can be affected at the same time, by means of the simple strategies comparable to dose titration as shown in this study.

In our experiments, perfusion (convective transport of O2) appears to contribute to O2 transport more than the bulk increase in O2-carrying capacity. The PBH concentration of 1.2 g/dl in plasma sustained MAP without significant arteriolar vasoconstriction, which increases arteriolar blood flow allowing more O2 to arrive to the microcirculation. In flowing arterioles, an increase in PO2 translates into more O2 available for the tissues. As anemic blood flows through arterioles, erythrocytes migrate away from the vessel wall, forming a cell-rich "core" region and a cell-depleted layer near the wall. Based on this model, acellular Hb increases axial convection and radial diffusion of O2 (20, 21). The Hb contained in RBCs is not subjected to molecular diffusion, and its O2 concentration is in equilibrium with the local O2 tension (20). In the 1960s, in vitro studies showed that nitrogen moving through Hb solutions diffused in proportion to its pressure differential, whereas O2 was transported by passive diffusion (proportion to its partial pressure) and diffusion of Hb-saturated O2 molecules (22, 30). As long as O2 is consumed outside the vessel wall, the diffusion distance is the same for molecular O2 and acellular oxy-Hb from the center of the vessel to its site of consumption. However, the flux of free O2 is a linear function of the O2 concentration, whereas the flux of oxy-Hb is a nonlinear function of the oxy-Hb concentration gradient, determined by the shape and position of the O2 equilibrium curve (i.e., a property of the Hb molecule). According to the Stokes-Einstein relation, facilitated diffusion of O2 is a function of the size of the oxy-Hb molecule and the viscosity of the Hb solution (22, 30). Wittenberg (30) showed that O2 flux through solutions of different-sized Hb molecules varied inversely with Hb size. Thus the flux of O2 can be controlled by changing the oxy-Hb diffusion constant, O2 equilibrium curve, and Hb saturation gradient.

An important result found in this study was that a moderate increase in O2-carrying capacity (PBH4) increased tissue PO2 to 23.5 ± 2.4 mmHg, 1.8 mmHg higher than the tissue PO2 at nonhemodiluted conditions for this tissue (21.7 ± 3.5 mmHg). Other studies have reported similar results. Standl et al. (25) subjected dogs to isovolemic hemodilution with 6% hetastarch, reducing their Hct to 10%, then augmenting their Hb level with PBH. They found that mean tissue PO2 measured with a needle electrode increased after transfusion compared with nontreated animals. This phenomenon was accompanied by a large increase in systemic O2 extraction, from baseline. In another study, the same investigators further reduced systemic Hct to 5% before infusion of PBH and found a similar increase in tissue PO2 (25, 26). These studies by Standl et al. introduced PBH in concentrations ranging from 1.5 to 7.2 g/dl, and, similar to the current study, the lower concentrations overcame the negative effects of vasoconstriction associated with this material and increased the O2 delivery. Additionally, according to a theoretical model proposed by Federspiel (11), free Hb in plasma facilitates O2 off-loading from the RBCs, an additional mechanism that further explains the increase in tissue O2 tension. The changes in O2 delivery did not change the amount of O2 released to the tissue (Fig. 5F), suggesting that the presence of PBH in the circulation did not affect O2 consumption and resulted in more O2 available to the tissue and, therefore, a higher tissue PO2.

In summary, PBH is a nonspecific glutaraldehyde polymerized Hb, with low O2 affinity (P50 of 54 mmHg) and cooperativity (Hill number = 1.2), resulting in a material that tends to easily release O2 (offload) (28). There is currently a tendency to consider low O2 affinity and reduced cooperativity as undesirable properties in HBOC formulations to replace or reinstate blood O2-carrying capacity during anemic conditions. The added inherent vasoactivity of PBH further complicates the scenario in which PBH can be optimally utilized. To date, it is impossible to fully replace RBC O2 transport with HBOC; however, enhancing O2 transport and offload of the remaining RBCs by additions of small amounts of acellular Hb solution could be extremely beneficial, in contrast to using them at high concentrations for the purpose of restoring the intrinsic O2-carrying capacity of blood.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Bioengineering Research Partnership Grant R24-HL-64395, Program project P01 HL-071064, and Grants R01-HL-62354, R01-HL-62318, and R01-HL-76182.


   ACKNOWLEDGMENTS
 
We thank Froilan P. Barra and Cynthia Walser for the surgical preparation of the animals.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Cabrales, La Jolla Bioengineering Institute, 505 Coast Boulevard South Suite #405, La Jolla, CA 92037 (e-mail: pcabrales{at}ucsd.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Altman DG, Bland JM. Statistics notes: how to randomise. Br Med J 319: 703–704, 1999.[Free Full Text]
  2. Cabrales P, Tsai AG. Plasma viscosity regulates systemic and microvascular perfusion during acute extreme anemic conditions. Am J Physiol Heart Circ Physiol 291: H2445–H2452, 2006.[Abstract/Free Full Text]
  3. Cabrales P, Tsai AG, Frangos JA, Briceno JC, Intaglietta M. Oxygen delivery and consumption in the microcirculation after extreme hemodilution with perfluorocarbons. Am J Physiol Heart Circ Physiol 287: H320–H330, 2004.[Abstract/Free Full Text]
  4. Cabrales P, Tsai AG, Frangos JA, Intaglietta M. Role of endothelial nitric oxide in microvascular oxygen delivery and consumption. Free Radic Biol Med 39: 1229–1237, 2005.[CrossRef][Web of Science][Medline]
  5. Cabrales P, Tsai AG, Intaglietta M. Deferoxamine lowers tissue damage after 80% exchange transfusion with polymerized hemoglobin. Antioxid Redox Signal 9: 375–384, 2007.[CrossRef][Web of Science][Medline]
  6. Cabrales P, Tsai AG, Intaglietta M. Increased tissue PO2 and decreased O2 delivery and consumption after 80% exchange transfusion with polymerized hemoglobin. Am J Physiol Heart Circ Physiol 287: H2825–H2833, 2004.[Abstract/Free Full Text]
  7. Cabrales P, Tsai AG, Intaglietta M. Microvascular pressure and functional capillary density in extreme hemodilution with low and high plasma viscosity expanders. Am J Physiol Heart Circ Physiol 287: H363–H373, 2004.[Abstract/Free Full Text]
  8. Cabrales P, Tsai AG, Intaglietta M. Nitric oxide regulation of microvascular oxygen exchange during hypoxia and hyperoxia. J Appl Physiol 100: 1181–1187, 2006.[Abstract/Free Full Text]
  9. Colantuoni A, Bertuglia S, Intaglietta M. Quantitation of rhythmic diameter changes in arterial microcirculation. Am J Physiol Heart Circ Physiol 246: H508–H517, 1984.[Abstract/Free Full Text]
  10. Endrich B, Asaishi K, Götz A, Messmer K. Technical report: A new chamber technique for microvascular studies in unanaesthetized hamsters. Res Exp Med 177: 125–134, 1980.[CrossRef][Medline]
  11. Federspiel WJ. Pulmonary diffusing capacity: implications of two-phase blood flow in capillaries. Respir Physiol 77: 119–134, 1989.[CrossRef][Web of Science][Medline]
  12. Guyton AC, Carrier O Jr, Walker JR. Evidence for tissue oxygen demand as the major factor causing autoregulation. Circ Res Suppl 15: 60–69, 1964.
  13. Guyton AC, Langston JB, Navar G. Theory for renal autoregulation by feedback at the juxtaglomerular apparatus. Circ Res Suppl 15: 187–197, 1964.
  14. Intaglietta M, Tompkins WR. Microvascular measurements by video image shearing and splitting. Microvasc Res 5: 309–312, 1973.[CrossRef][Web of Science][Medline]
  15. Kemming GI, Meisner FG, Meier J, Tillmanns J, Thein E, Eriskat J, Habler OP. Hyperoxic ventilation at the critical hematocrit: effects on myocardial perfusion and function. Acta Anaesthesiol Scand 48: 951–959, 2004.[CrossRef][Web of Science][Medline]
  16. Kerger H, Groth G, Kalenka A, Vajkoczy P, Tsai AG, Intaglietta M. PO2 measurements by phosphorescence quenching: characteristics and applications of an automated system. Microvasc Res 65: 32–38, 2003.[CrossRef][Web of Science][Medline]
  17. Lipowsky HH, Zweifach BW. Application of the "two-slit" photometric technique to the measurement of microvascular volumetric flow rates. Microvasc Res 15: 93–101, 1978.[CrossRef][Web of Science][Medline]
  18. Lowe KC, Farrell K, Ferguson EM, James V. Current perceived risks of transfusion in the UK and relevance to the future acceptance of blood substitutes. Artif Cells Blood Substit Immobil Biotechnol 29: 179–189, 2001.[CrossRef][Web of Science][Medline]
  19. Meier J, Kemming GI, Kisch-Wedel H, Wolkhammer S, Habler OP. Hyperoxic ventilation reduces 6-hour mortality at the critical hemoglobin concentration. Anesthesiology 100: 70–76, 2004.[CrossRef][Web of Science][Medline]
  20. Nair PK, Hellums JD, Olson JS. Prediction of oxygen transport rates in blood flowing in large capillaries. Microvasc Res 38: 269–285, 1989.[CrossRef][Web of Science][Medline]
  21. Page TC, Light WR, McKay CB, Hellums JD. Oxygen transport by erythrocyte/hemoglobin solution mixtures in an in vitro capillary as a model of hemoglobin-based oxygen carrier performance. Microvasc Res 55: 54–64, 1998.[CrossRef][Web of Science][Medline]
  22. Scholander P. Oxygen transport through hemoglobin solutions. Science 131: 585–590, 1960.[Free Full Text]
  23. Sellards A, Minot G. Injection of hemoglobin in man and its relation to blood destruction with special reference to anemias (Abstract). J Med Res 34: 469, 1916.
  24. Spahn DR, Smith LR, Veronee CD, McRae RL, Hu WC, Menius AJ, Lowe JE, Leone BJ. Acute isovolemic hemodilution and blood transfusion. Effects on regional function and metabolism in myocardium with compromised coronary blood flow. J Thorac Cardiovasc Surg 105: 694–704, 1993.[Abstract]
  25. Standl T, Horn P, Wilhelm S, Greim C, Freitag M, Freitag U, Sputtek A, Jacobs E, Schulte am Esch J. Bovine haemoglobin is more potent than autologous red blood cells in restoring muscular tissue oxygenation after profound isovolaemic haemodilution in dogs. Can J Anaesth 43: 714–723, 1996.[Web of Science][Medline]
  26. Standl T, Wilhelm S, Horn EP, Burmeister M, Gundlach M, Schulte am Esch J. Preoperative hemodilution with bovine hemoglobin Acute hemodynamic effects in liver surgery patients. Anaesthesist 46: 763–770, 1997.[CrossRef][Web of Science][Medline]
  27. Tsai AG, Friesenecker B, McCarthy M, Sakai H, Intaglietta M. Plasma viscosity regulates capillary perfusion during extreme hemodilution in hamster skin fold model. Am J Physiol Heart Circ Physiol 275: H2170–H2180, 1998.[Abstract/Free Full Text]
  28. Tsai AG, Johnson PC, Intaglietta M. Oxygen gradients in the microcirculation. Physiol Rev 83: 933–963, 2003.[Abstract/Free Full Text]
  29. Winslow RM. Oxygen transport agents: a new approach to red blood cell alternatives. TATM 5: 498–504, 2004.
  30. Wittenberg JB. The molecular mechanism of hemoglobin-facilitated oxygen diffusion. J Biol Chem 241: 104–114, 1966.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/5/H2212    most recent
00751.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cabrales, P.
Right arrow Articles by Intaglietta, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cabrales, P.
Right arrow Articles by Intaglietta, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.