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Am J Physiol Heart Circ Physiol 276: H553-H562, 1999;
0363-6135/99 $5.00
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Vol. 276, Issue 2, H553-H562, February 1999

Microvascular responses to hemodilution with Hb vesicles as red blood cell substitutes: influence of O2 affinity

Hiromi Sakai1,2, Amy G. Tsai1, Ronald J. Rohlfs1, Hiroyuki Hara1,2, Shinji Takeoka2, Eishun Tsuchida2, and Marcos Intaglietta1

1 Departments of Bioengineering and Medicine, University of California, San Diego, La Jolla, California, 92093-0412; and 2 Department of Polymer Chemistry, Advanced Research Institute for Science and Engineering, Waseda University, Tokyo 169-8555, Japan


    ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Phospholipid vesicles encapsulating purified hemoglobin (HbV) were developed to provide O2-carrying capacity to plasma expanders. Microvascular perfusion was determined for HbV with different O2 affinity (P50 = 9, 16, and 30 mmHg) prepared by coencapsulating pyridoxal 5'-phosphate (PLP) at the molar ratios of [PLP]/[Hb] = 0, 0.5, and 3, respectively (cf. hamster blood, P50: 28 mmHg), and suspended in 8 g/dl human serum albumin (HSA). Eighty percent of the red blood cell (RBC) mass of conscious Syrian golden hamsters fitted with dorsal skinfold windows was substituted with either of the HbV-HSA suspensions, washed hamster RBC suspended in HSA (RBC-HSA), and HSA alone. All three HbV-HSA groups and RBC-HSA groups showed stable blood pressure and heart rate, which could not be sustained with HSA alone. Only the HbV (P50 = 9)-HSA group showed an increase in arterial O2 tension (89.8 ± 14.7 mmHg, baseline 58.4 ± 4.0 mmHg) because of hyperventilation, and microvascular perfusion was decreased, indicating that facilitated O2 unloading of HbV by decreasing the O2 affinity (increasing P50) with PLP as an allosteric effector is important. Microvascular perfusion and microvascular and interstitial O2 tensions in the HbV (P50 = 16 and 30)-HSA groups were significantly higher than those in the HSA group. The O2 release rate from the HbV was 18-32 s-1 vs. 4.4 s-1 for RBC. Functional capillary density was improved from 17 to 41% on average by decreasing P50 from 30 to 16 mmHg, which appears to be an optimal value for the P50 in this system.

oxygen carrier; microcirculation; liposome; oxygen dissociation curve; autoregulation


    INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

PHOSPHOLIPID VESICLES encapsulating concentrated hemoglobin (Hb vesicles, HbV) have the potential of becoming industrially produced red blood cell (RBC) substitutes. They most closely reproduce the characteristics of natural blood, including the RBC membrane function, by physically preventing direct contact of Hb with the cellular components of circulation (3, 19-21, 23, 24, 33). The desirability of this barrier function is evident in considering the side effects found in the use of acellular Hb solutions such as chemically modified Hb and recombinant Hb, which are now in clinical trials (34). Hb encapsulation potentially shields the microcirculation from the biological activity or toxicity of Hb and Hb-related products such as methemoglobin (metHb) and released heme. The principal systemic side effect consistently reported in the administration of acellular Hb solutions is a pressor response that has been attributed to the nitric oxide-scavenging effect of Hb due to the intrinsic high affinity of nitric oxide to Hb, a process presumed to lead to vasoconstriction (11, 18, 26). Conversely, nitric oxide-related vasoconstriction by HbV has not been observed (19).

O2 affinity of blood is a crucial factor in determining O2 delivery and unloading to tissues. There have been numerous theoretical analyses on the relationship between the modification of O2 affinity (P50) of RBC and resulting hemodynamic changes. Shifting the O2 dissociation curve to the right (higher P50) facilitates O2 unloading to the tissue, reduces cardiac output, and increases vascular resistance in the absence of changes in O2 consumption or blood pressure (15, 29). On the other hand, shifting the O2 dissociation curve to the left (lower P50) is advantageous only during severe hypoxia, because the O2 delivery to tissues is enhanced (6, 27, 27a, 30, 35).

In terms of RBC substitutes, it has been assumed that their O2 affinity should be the same or lower than that of blood to increase the arteriovenous difference in O2 saturation and facilitate O2 unloading. The relationship between the O2 affinity of RBC substitutes and systemic or microvascular perfusion has not been presently reported. The O2 affinity of HbV can be controlled (P50 = 5-150 mmHg) by selecting the appropriate amount of coencapsulated allosteric effector [pyridoxal 5'-phosphate (PLP), inositol hexaphosphate, Cl-, H+, etc.] without changing other physical properties (33). We prepared three kinds of HbV suspended in 8 g/dl human serum albumin (HSA) solution with different P50 values (9, 16, and 30 mmHg) and evaluated the effect on subcutaneous microvascular responses to severe hemodilution using conscious hamsters fitted with a dorsal skinfold chamber (8, 9, 12, 13, 24).


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Preparation of Hb vesicles with different O2 affinities. HbVs were prepared as previously reported in the literature (21, 23). The encapsulated hemoglobin (38 g/dl) contained PLP as an allosteric effector. The inner concentrations of PLP were 0, 6, and 18 mM for the P50 of 9, 16, and 30 mmHg, respectively. The surface of HbV was modified with polyethyleneglycol (PEG, mol wt = 5,000) by mixing the HbV suspension with a saline solution of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-(polyethyleneglycol). HbV was ultracentrifuged and redispersed in a 8 g/dl HSA solution prepared from albumin-25% (Bayer, Germany) and saline. In our previous study, HbV was suspended in 5 g/dl HSA (24); however, we changed to 8 g/dl HSA because it showed better microvascular perfusion (i.e., increased RBC velocity and functional capillary density) than 5 g/dl HSA. The suspension was then filtered through sterilizable filters (pore size: 0.45 µm), and the Hb concentration was regulated to 10 g/dl using the HSA solution to obtain HbV-HSA suspensions with different P50 values. The characteristics of PEG-modified HbV-HSA suspensions are listed in Table 1, with all parameters being almost identical except for O2 affinity.

                              
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  		Table 1.   Characteristics of polyethyleneglycol-modified HbVs suspended in 8% HSA with different oxygen affinity

Measurements of O2 affinity and rate of O2 release from HbV. O2 affinity (P50) and the Hill number of each HbV and RBC were calculated from O2 dissociation curves measured with a Hemox Analyzer (TCS-Medical Products) at 37°C. The kinetics of O2 release from HbV were measured by conventional rapid mixing techniques using a stopped-flow spectrophotometer (model 17 MV, Applied Photophysics, London, UK). Suspensions of air-equilibrated HbV were mixed against anaerobic solutions of dithionate, and the conversion of oxyhemoglobin to deoxyhemoglobin was monitored by the absorbance changes at 415 and 436 nm. HbV samples suspended in aerobic 50 mM phosphate-buffered saline (pH 7.4, room temperature) and 50 mM sodium dithionite were dissolved in anaerobic 50 mM phosphate-buffered saline (31). Sodium dithionite reacts with O2 to diminish the dissolved O2, thus HbO2 converts to deoxyhemoglobin. The absorption decrease at 415 nm and the increase at 436 nm were monitored during deoxygenation. Four curves were obtained for each wavelength, and the rates were averaged.

Preparation of washed hamster RBC suspended in HSA. Hamster RBC suspended in HSA (RBC-HSA) were used as a reference to compare the effectiveness of HbV and to demonstrate the influence of the exchange transfusion procedure. Syrian golden hamsters (body weight of 140-150 g) were anesthetized with pentobarbital sodium (100 mg/kg body wt). A polyethylene catheter (PE-10) was implanted in the carotid artery, and blood was withdrawn with heparinized syringes, while 8 g/dl HSA solution was infused alternately by exchange transfusion. The collected blood was centrifuged to obtain an RBC concentrate. This concentrate was washed twice to remove plasma components and buffy coat by redispersion in HSA and centrifugation at 3,000 g for 3.5 min. The Hb concentration was adjusted to 10 g/dl, which was equivalent to the concentration of HbV-HSA.

Animal model and preparation. Experiments were carried out in 50 male Syrian golden hamsters of 72 ± 8 g body wt (Simonsen, Gilroy, CA). With each rat under intraperitoneal pentobarbital sodium anesthesia (100 mg/kg body wt), the dorsal skinfold consisting of two layers of skin and muscle was fitted with two titanium frames with a 15-mm circular opening and surgically installed (12, 13, 24). Layers of skin muscle were separated from the subcutaneous tissue and removed until a thin monolayer of muscle and one layer of intact skin remained. A cover glass (diameter, 12 mm) was held by one frame on the exposed tissue allowing intravital observation of the microvasculature and tissues.

Polyethylene tubes (PE-10, 1 cm), which were connected to PE-50 (25 cm) via silicone elastomer medical tubes (4 cm, Technical Products), were implanted in the jugular vein and the carotid artery. They were passed from the ventral to the dorsal side of the neck and exteriorized through the skin at the base of the chamber. Patency of the catheters was ensured by filling with heparinized saline (40 IU/ml).

Microvascular observations of the awake and unanesthetized hamsters were performed at least 3 days after chamber implantation to mitigate postsurgical trauma. We used conscious hamsters because anesthesia alters microvascular perfusion, which depends on the deepness of anesthesia (12). During the measurements, the animals were placed in a perforated plastic tube (inner diameter, 3.8 cm; length, 17 cm), from which the window chamber protrudes, to minimize animal movement without impeding respiration.

A preparation was considered suitable for experimentation if microscopic examination of the window chamber met the following criteria: 1) no sign of bleeding and/or edema; 2) systemic mean arterial pressure (MAP) was >80 mmHg; and 3) heart rate was above 320 beats/min.

All animal studies were approved by the Animal Subject Committee of University of California, San Diego, and the National Institutes of Health Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23, Revised 1985] principles have been observed.

Experimental procedure. Isovolemic hemodilution with the experimental fluids was performed by the simultaneous withdrawal of blood from the arterial catheter and infusion of the test substitutes into the venous catheter at a rate of 0.3 ml/min as described previously (24).

Microhemodynamic and systemic parameters were evaluated at 0, 30, 60, and 80% levels of exchange. Thirty-three hamsters were exchange transfused in five groups with PEG-modified HbV-HSA with P50 = 9 mmHg (n = 6), 16 mmHg (n = 7), and 30 mmHg (n = 7), and washed hamster RBC-HSA (n = 6), and HSA alone (n = 7). The in situ microcirculation of the skinfold chamber was observed using a video-microscope system. After the blood substitution, a bolus intravenous injection of palladium-porphyrin bound to bovine albumin solution (7.6 wt%, 0.1 ml) was injected to measure the O2 tensions (PO2) in vessels and interstitium (12, 13) (see Determination of microvascular and interstitial O2 tensions). Seventeen hamsters were used for baseline PO2 measurement.

Microhemodynamic analysis. Microvessels in the subcutaneous tissue and the skeletal skin muscle were observed with an inverted microscope and by the transillumination technique. Microvessels were classified according to their position within the microvascular network. Arteriolar microvessels were grouped into large feeding arterioles (A1; diameter, 57 ± 14 µm), small arcading arterioles (A2, 25 ± 8 µm), transverse arterioles (A3, 10 ± 2 µm), and terminal arterioles (A4, 9 ± 2 µm). Venules were classified as small collecting venules (VC, 28 ± 6 µm) and large venules (VL, 80 ± 15 µm). The microvessels selected for measurements were chosen for their optical clarity and not by the nature of the flow. Capillaries and tissue segments selected for measurements were supplied and drained by the arterioles and venules of a functional microvascular unit. These microvessels and capillaries were sketched in advance to plan the sequence of measurements.

Microvascular diameter and RBC velocity were analyzed on-line in arterioles and venules (8, 24). Vessel diameter was measured with an image-shearing system, whereas RBC velocity was analyzed by photodiodes and the cross-correlation technique. Blood flow rates (Q) were calculated using the equation
<A><AC>Q</AC><AC>˙</AC></A> = (RBC velocity/R<SUB>v</SUB>) ⋅ &pgr; ⋅ (diameter/2)<SUP>2</SUP> (1)
where Rv represents the ratio of velocity in the middle of vessels to whole blood velocity based on the data in the glass tubes. Rv at 1.6 was used for A1, A2, VC, and VL, and Rv at 1.3 was used for A3 and A4.

Functional capillary density was analyzed on-line by counting the number of capillaries with RBC flow stemming from one A3 arteriole, usually 40-80 capillaries, and expressed as a percentage of the basal value.

Characterization of systemic conditions. Blood samples were collected in heparinized microtubes for the hematocrit and blood gas analyses. A pH/blood gas analyzer (Ciba Corning 238, Ciba Corning Diagnostic, Pleasanton, CA) was used for analysis of arterial blood O2 tension (PaO2), arterial blood carbon dioxide tension (PaCO2), pH, and base excess. An analogue recording system (Beckman R611, Beckman Instruments, Schiller Park, IL) was used for continuous monitoring of MAP and heart rate.

Determination of microvascular and interstitial O2 tensions. Subcutaneous microvascular and interstitial PO2 were determined with the O2-dependent quenching of phosphorescence emitted by palladium-meso-tetra(4-carboxyphenyl)porphyrin bound to serum albumin after pulsed light excitation (8, 9, 12, 13). The method allows noninvasive assessment of intravascular PO2 and determination of interstitial oxygenation, because intravascularly injected porphyrin-albumin complexes extravasate into the interstitium over time. PO2 can be obtained using the Stern-Volmer equation from the phosphorescence lifetimes.

Percentage of microvascular O2 consumption after 80% level of exchange (VmO2) in comparison with baseline was calculated using the equation
<A><AC>V</AC><AC>˙</AC></A><SUB>mO<SUB>2</SUB></SUB> = <FR><NU><AR><R><C><A><AC>Q</AC><AC>˙</AC></A><SUB>av</SUB> × {[Hb]<SUB>RBC</SUB> × (S<SUB>A<SUB>1</SUB></SUB>O<SUB>2</SUB> − S<SUB>V<SUB>L</SUB></SUB>O<SUB>2</SUB>) </C></R><R><C>+ [Hb]<SUB>Hb V</SUB> × (S′<SUB>A<SUB>1</SUB></SUB>O<SUB>2</SUB> − S′<SUB>V<SUB>L</SUB></SUB>O<SUB>2</SUB>)}</C></R></AR></NU><DE>14 × 18</DE></FR> (2)
where Qav is the averaged percentage of blood flows in A1 and VL relative to baseline. [Hb]RBC and [Hb]HbV are Hb concentrations of RBC and HbV, respectively, which were estimated from the hematocrit. SA1O2 and S'A1O2 are O2 saturations in A1 of RBC and HbV, respectively, and SVLO2 and S'VLO2 are those in VL. They were estimated from the O2 dissociation curves of HbVs and RBC in Fig. 1 and microvascular O2 tensions. The initial Hb concentration and arteriovenous difference in the microvasculature (SA1O2 - SVLO2) before the blood exchange were estimated to be 14 g/dl and 18%, respectively.


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  		Fig. 1.   O2 dissociation curves of 3 hemoglobin vesicles (HbV) with different O2 affinities measured with an Hemox analyzer at 37°C and hamster red blood cells (RBC) cited from Stein and Ellsworth (27a).

Data analysis. Data were analyzed using ANOVA followed by Fisher's protected least significant difference test between the groups. Paired t-test was used for the time-dependent comparisons with the baseline values in each group. The level of confidence was placed at 95% for all the experiments.


    RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

O2 affinities and O2 release rates of HbV. The value of P50 (PO2 at which Hb saturates with half O2) of HbV without PLP as an allosteric effector showed 9 mmHg, and it increased to 16 and 30 mmHg by coencapsulating PLP at the ratios of PLP/Hb = 0.5 and 3, respectively (Fig. 1). O2 transport efficiencies (OTE; difference in O2 saturation between arterial and venous O2 tension) were 5, 16, and 32%, when calculated using the human arteriovenous difference at P50 = 9, 16, and 30 mmHg, respectively, and 4, 19, and 36%, respectively, when calculated using the hamster arteriovenous difference. Even though the P50 of HbV (P50 = 30) was close to that of hamster RBC (28 mmHg), its Hill number (indicator of subunits cooperativity) was smaller than that of hamster RBC. Therefore, the slope of HbV (P50 = 30) is steeper at a PO2 of 60 mmHg, and the trend reverses at ~30 mmHg.

The rates of O2 release of three HbVs in the presence of excess sodium dithionite in the outer media were 18, 27, and 32 s-1 at P50 = 9, 16, and 30 mmHg, respectively (Fig. 2). These rates were much slower than the rate of purified small arteries (HbAo; 84 s-1). However, these are much faster than RBC (2.0-9.1 s-1) reported by Vandegriff and Olson (31).


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  		Fig. 2.   Time courses of O2 release by RBC, HbVs, and HbAo, measured with a stopped flow method, mixing an air-equilibrated Hb containing sample (10 µM) and deoxygenated sodium dithionate solution (50 mM), room tempertature, in 50 mM phosphate-buffered saline (pH 7.4). Inset, from top to bottom: RBC, HbV (P50 = 9, 16, and 30 mmHg) and HbAo. Curve of human RBC was cited from Vandegriff and Olson (31). kr, O2 release rate.

Changes in systemic parameters during hemodilution. Hematocrit before the exchange transfusion was 47-50% on the average, and it was reduced to ~7% after 80% level of exchange for all the groups except the RBC-HSA group, which was 31% (Table 2). This was due to the infusion of RBC suspension with a hematocrit of ~30%. The actual levels of blood exchange calculated from the hematocrit were ~32, 64, and 85% for the theoretical values of 30, 60, and 80%, respectively.

                              
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  		Table 2.   Systemic hematocrit and blood gas parameters during hemodilution with HSA, HbV/HSAs, or hamster RBC/HSA

The HSA and HbV (P50 = 9)-HSA groups had increased PaO2. The HSA group showed a dramatic change from 61.6 ± 9.4 mmHg as baseline to 117.3 ± 4.6 mmHg. The HbV (P50 = 9)-HSA group increased from 58.4 ± 4.0 mmHg to 89.8 ± 14.7 mmHg. However, the other two groups HbV (P50 = 16 and 30)-HSA groups showed stable values, and they were significantly lower than the others.

The HSA and HbV (P50 = 9)-HSA groups showed a significant drop in PaCO2 from 56.0 ± 3.2 and 60.3 ± 4.0 mmHg at baseline to 37.6 ± 7.1 and 47.2 ± 11.0 mmHg at 80% level of exchange, respectively. The other three groups, HbV (P50 = 16 and 30)-HSA, and RBC/HSA had stable values that were significantly higher than the HSA group.

The HSA group exhibited a significant increase in pH from 7.390 ± 0.029 at baseline to 7.448 ± 0.069 at 80% level of exchange because of respiratory alkalosis. The RBC-HSA group also showed a drop in pH, whereas it was significantly higher than the HSA group.

The HSA group showed a significant drop in base excess to -1.65 ± 0.21 mmol/l (baseline, 5.10 ± 0.85 mmol/l), indicating severe anemia. The other four groups also showed the drops in base excess, whereas they were significantly higher than the HSA group.

As for the systemic hemodynamics, all the HbV-HSA groups showed slight increases in MAP (Fig. 3). The HSA group started to show a significant decrease at 60% level of exchange and dropped to 66.8 ± 17.1 mmHg (baseline, 103.0 ± 8.3 mmHg) at 80% exchange. The other four groups receiving O2-carrying fluids showed higher values that were stable even after the completion of the blood exchange.


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  		Fig. 3.   Changes in mean arterial pressure (MAP; A) and heart rate (B) during hemodilution with HbV-human serum albumin (HSA), HSA, and washed RBC-HSA. Values are means ± SD. * Significantly different from baseline (P < 0.05).

Heart rate of all the groups maintained the normal values except the HSA group, which showed an increase to 509 ± 74 beats/min at 60% level of exchange and then a decrease to 309 ± 174 beats/min at 80% level of exchange (baseline, 448 ± 69 beats/min) because of cardiovascular insufficiency consequent to anemia.

Microhemodynamic responses to hemodilution. Basal values for the microvessel diameters and blood flow rates are shown in Table 3. There is no significant difference between the groups.

                              
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  		Table 3.   Basal values for the diameters and blood flow rates of arterioles and venules

Venules of the HSA group constricted up to the 80% level of exchange (VL, -26% on the average in comparison with baseline; VC, -7%) (Fig. 4), whereas the arterioles tended to dilate. These phenomena may be related to blood redistribution from the skin to the vital organs as anemia progressed. The RBC-HSA group showed some diameter changes, but they were not consistent. The three HbV-HSA groups showed rather stable values.


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  		Fig. 4.   Diameter changes of arterioles (A1-A4) and collecting and large venules (VC and VL, respectively) of skinfold preparation during hemodilution. Values are means ± SD. * Significantly different from baseline (P < 0.05); # significantly different between indicated groups (P < 0.05).

The HSA group showed higher rates of blood flow in comparison with baseline in A2 (+9%) and A3 (+33%) at 30% level of exchange and in A1 (+18%), A3 (+17%), and A4 (+40%) at 60% level of exchange (Fig. 5). This is a typical phenomenon for hemodilution with plasma expanders (17). However, the HSA group had a significant drop to nearly zero at the final exchange. The other four groups showed significantly higher rates of blood flow at 80% level of exchange. The RBC-HSA group maintained the flow rates in A1, A2, and VL and showed significantly higher rates at 60% level of exchange in VC (+91%). The HbV-HSA groups showed lower values than the RBC-HSA group; however, the HbV (P50 = 16)-HSA group showed nonsignificantly the highest values among the three in A1, A2, A3, VC, and VL. Flow in the HbV (P50 = 9)-HSA group dropped significantly at the first 30% level of exchange and maintained the lowest value among the three.


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  		Fig. 5.   Changes in flow rates in arterioles (A1-A4) and venules (VC and VL) of skinfold preparation during hemodilution. Values are means ± SD. * Significantly different from baseline (P < 0.05); # significantly different between indicated groups (P < 0.05).

Functional capillary density of the RBC-HSA group was maintained at the highest value throughout the hemodilution (80 ± 28% at 80% level of exchange) (Fig. 6). The HSA group maintained high values up to the 60% level of exchange; however, it dropped to 1.4 ± 2.3% at the final exchange. The three HbV-HSA groups showed significantly lower values than the RBC-HSA group; however, they were all higher than the HSA group at the 80% level of exchange. The HbV (P50 = 16)-HSA group showed the highest density among the three. Because functional capillary density was measured by counting the number of capillaries where there was flow of RBCs, the values for the HSA and HbV-HSA groups might be underestimated because of plasma skimming.


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  		Fig. 6.   Changes in functional capillary density during hemodilution. Values are means ± SD. * Significantly different from baseline (P < 0.05); # significantly different between indicated groups (P < 0.05).

Microvascular and interstitial O2 tensions. Normally A1 had 51.1 ± 6.3 mmHg in PO2, decreasing to 39.3 ± 4.8 mmHg in A4 arterioles (Fig. 7). This reduction is associated with the diffusion of O2 from these arterioles (4, 9, 28). After perfusion through the capillaries, the PO2 in venules increased from 33.8 ± 9.8 mmHg in VC to 35.6 ± 5.8 mmHg in VL because of the presence of the O2 shunt. The PO2 values of the HSA group were consistently very low (<23.9 ± 9.7 mmHg). The other four groups maintained significantly higher values than the HSA group, and the RBC-HSA group had PO2 values that were close to baseline. The three HbV-HSA groups were lower than the RBC-HSA group; however, the HbV (P50 = 16 and 30)-HSA groups showed significantly higher values than the HbV (P50 = 9)-HSA group in A1, A2, and A4. Interstitial PO2 is slightly higher for HbV (P50 = 16)-HSA than HbV (P50 = 30)-HSA.


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  		Fig. 7.   O2 tensions of microvasculature (A1-A4, VC, and VL) and interstitium after 80% exchange and baseline value. Values are means ± SD. * Significantly different from baseline (P < 0.05).

Relative microvascular O2 consumption. Relative O2 consumption in the microvasculature after exchange (VmO2) in comparison with the baseline can be estimated from the change in blood flow, Hb concentration, and arteriovenous (A1-VL) difference. As shown in Fig. 8, the HSA group showed 7.4% of VmO2 relative to baseline. Other groups showed significantly higher values than the HSA group, although the HbV (P50 = 9)-HSA group showed significantly lower VmO2 than the baseline (19.0%). The RBC-HSA group maintained the highest level of VmO2 (101.0%). The HbV (P50 = 16 and 30)-HSA groups showed almost the same values on the average (79.7 and 80.7%, respectively). They were lower than the RBC-HSA group without significant difference. The A1-VL differences in O2 saturation of RBC (SA1O2 - SVLO2) were 37, 47, 44, 58, 30, and 18% for the groups of HSA, HbV (P50 = 9, 16, and 30)-HSA, RBC-HSA, and baseline, respectively. Those differences of HbV (S'A1 O2 - S'VLO2) were 38, 32, and 51% for HbV-HSA (P50 = 9, 16, and 30, respectively). The HbV-HSA (P50 = 30) showed the highest A1-VL differences in O2 saturation for both RBC and HbV.


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  		Fig. 8.   Relative O2 consumptions (VmO2) of microvasculature after 80% blood exchange in comparison with baseline. Values are means ± SE. * Significantly different from baseline (P < 0.05).


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Our results show that all three HbV-HSA groups with different P50 values showed similar stable systemic hemodynamics during severe hemodilution, which could not be sustained by HSA alone; however, there were significant differences in blood gas and microhemodynamic parameters. This becomes apparent when severe hemodilution is performed to hematocrit <10%. This level of blood exchange is necessary for a testing of RBC substitutes in terms of safety and efficacy (1, 3, 24), because an exchange of up to 60% with non-O2-carrying HSA does not show significant differences from normal microhemodynamic parameters. Among the three HbV groups, HbV (P50 = 16 and 30)-HSA showed significantly improved microvascular perfusion than the HbV (P50 = 9)-HSA, which emphasizes the importance of facilitated O2 unloading of HbV by decreasing the O2 affinity with PLP as an allosteric effector. Although the HbV (P50 = 9)-HSA group maintained stable blood pressure and heart rate, the increase in PaO2 was remarkable. This is due to hyperventilation, suggesting the decreased O2 off-loading from the HbV with high O2 affinity. Arteriovenous difference was increased by the lowered O2 tension in VL (38%; baseline, 18%); however, O2 consumption in the subcutaneous microvasculature was diminished mainly as a consequence of the significantly decreased blood flow rates.

The HbV (P50 = 16 and 30)-HSA groups maintained values comparable with the RBC-HSA group up to the 60% level of exchange, suggesting an improved O2 supply. Even though the microvascular perfusion of the HbV (P50 = 16 and 30)-HSA groups were inferior to the RBC-HSA group at 80% level of exchange, the HbV (P50 = 16)-HSA group showed relatively higher functional capillary density and flow rates than the P50 = 30 group. This corresponds to the result of Woodson and Auerbach (35), who found increased blood flow in the heart, brain, and spleen without changes in cardiac output and blood pressure when O2 affinity of rat RBCs was increased to a P50 = 17 mmHg from the normally 38 mmHg. They concluded that this is a compensatory response to decreased O2 delivery. It was shown previously in our model that most of the O2 is diffusively supplied primarily from arterioles rather than capillaries in normal condition, as evidenced by the fall in O2 tension from A1 to A4 arterioles in Fig. 7 (9). Because functional capillary density may be related to mechanisms in addition to O2 delivery, an improvement of this parameter may lead to enhanced tissue viability.

There was not enough vasoconstriction for the HbV-HSA groups to explain the reduced blood flow rates in comparison with the RBC-HSA group. Subcutaneous blood flow is probably regulated in part at the level of microvasculature under the observation and by thoracodorsal arteries or larger arteries not visible in the preparation (22). Normally, the low O2 solubility in plasma creates a major barrier to the diffusion of O2 in the plasma space between RBCs and vascular walls. It has been speculated that exposure of arteries or arterioles to high O2 tension induced autoregulatory limitation of O2 delivery by restricting flow with decreased endothelial prostaglandin synthesis, thus increasing peripheral vascular resistance (2, 7, 10, 16). The hypothesis has been suggested that acellular RBC substitutes (Hb solution) produce a higher O2 availability because of the homogeneously dissolved Hb, leading to a microcirculation-limited O2 delivery due to vasoconstriction (9, 32). HbVs may have the same effect because they are much smaller than RBCs and are dispersed relatively homogeneously throughout the plasma (24). As shown in Fig. 2, O2 release from the acellular Hbs is faster than the release from HbVs and RBCs at the same heme concentration and same O2 affinity, because O2 must diffuse through the viscous Hb solutions in the vesicles to the outside and through the solvent layer surrounding each HbV particle and RBC. O2 release from the HbV is much faster than that from RBC because particle size is smaller (0.25 µm vs. 8 µm), providing a much smaller surface area-to-volume ratio for diffusion (32). This may explain the higher functional capillary densities and blood flow rates for HbV (P50 = 16)-HSA vs. HbV (P50 = 30)-HSA. However, we could not obtain better results with HbV (P50 = 9)-HSA even though its O2 release rate is slowest and the closest to that of RBC. All known Hb-related blood substitutes appear to have faster O2 release than HbVs (P50 = 9). Given these considerations, the amount of O2 unloaded (arteriovenous difference or O2 transporting efficiency) estimated from the O2 dissociation curve may be crucial.

The different shapes of O2 dissociation curves (Fig. 1) can also explain our observations. Even though the O2 affinity of HbV (P50 = 30) is close to that of the hamster RBC (P50 = 28), the Hill number of HbV is smaller than that of RBC. Comparing the slopes of the curves at around P50, RBC is steeper than HbV (P50 = 30). Conversely, in the range of 60-50 mmHg, which corresponds to O2 tensions at the aorta (PaO2) and the entrance of the microvasculature (A1), HbV (P50 = 30) is steeper than the hamster RBC. This indicates that HbV (P50 = 30) releases a larger amount of O2 in large arteries than RBC does, which may induce autoregulatory response to overabundant O2 and resulting in constriction of upstream arteries, which are larger than A1. HbV (P50 = 16), which shows a gentler slope, may induce less vasoconstriction. In the case of HbV (P50 = 9), the absolute amount of unloaded O2 may be crucial. Considering this hypothesis, the better slope of HbV at around 50 mmHg should be identical to that of RBC, and at this condition P50 should be 22 mmHg because the Hill number of HbV is smaller than that of RBC. However, it is impossible to regulate both the O2 affinity and O2-releasing rate to be identical to those of RBC, which is a limitation due to the small size of HbV.

Other mechanisms may also contribute to the reduction in blood flow relative to baseline, such as influences of the complement activation and active oxygen production, which may alter the microvascular perfusion.

In conclusion, the present experiments with three kinds of HbVs with different P50 and same other physical properties show that microvascular perfusion and O2 tensions of the three HbV-HSA groups were much improved in comparison with the HSA group; these parameters were slightly improved by decreasing P50 from 30 to 16 mmHg, which appears to be an optimal value for P50 in this system. Further increasing P50 to 9 mmHg reversed the trend, indicating that facilitated O2 unloading of HbV by decreasing the O2 affinity with PLP as an allosteric effector is important. This result indicates that the optimal O2 dissociation curve of HbV in the normoxic condition, from the view point of microvascular perfusion, may be left shifted relative to blood.


    ACKNOWLEDGEMENTS

The authors gratefully acknowledge Dr. Robert M. Winslow for supervising the study of blood substitutes. The authors also thank Dr. K. D. Vandegriff for the discussion on the stopped flow analysis, Dr. M. McCarthy for the oncotic pressure and O2 capacity measurements, and Dr. S. I. Park and T. Kose, M. Aburatani, and K. Hamazaki for the HbV preparation.


    FOOTNOTES

This work has been supported in part by National Heart, Lung, and Blood Institute Program Project HL-48018 and Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan (Grant 07508005). H. Sakai was an overseas research fellow of the Japan Society for the Promotion of Science.

Addresses for reprint requests and correspondence: M. Intaglietta, Dept. of Bioengineering, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0412; E. Tsuchida, Dept. of Polymer Chemistry, Advanced Research Institute for Science and Engineering, Waseda Univ., Tokyo 169-8555, Japan.

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. §1734 solely to indicate this fact.

Received 17 February 1998; accepted in final form 13 October 1998.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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Am J Physiol Heart Circ Physiol 276(2):H553-H562
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


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