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1Department of Bioengineering, University of California-San Diego, and 2La Jolla Bioengineering Institute, La Jolla, California; and 3Advanced Research Institute for Science and Engineering, Waseda University, Tokyo, Japan
Submitted 1 October 2004 ; accepted in final form 18 November 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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oxygen-carrying capacity; blood substitutes; tissue oxygen; hemoglobin oxygen affinity
Recent developments in the field of oxygen-carrying plasma expanders (OCPE) based on molecular Hb solutions reported by Tsai et al. (22) show that the addition of comparatively small amounts of a significantly left-shifted polyethylene glycol-conjugated oxygen carrier (P50 
5 mmHg) to blood in extreme hemodilution leads to baseline microvascular and systemic conditions. This result could not be obtained in identical extreme hemodilution experiments with the use of a right-shifted molecular Hb solution at a considerably higher concentration (19).
Extreme hemodilution in the hamster window chamber model to a hematocrit (Hct) level of 11% is a powerful tool to test the efficacy of OCPEs in restoring microvascular function and systemic conditions. This Hct is below the threshold at which the organism becomes oxygen supply limited (5, 22, 23). In this scenario, the effects of a blood substitute became magnified upon introduction into the circulation. Furthermore, by encapsulating Hb, a phospholipid vesicle eliminates the problem of Hb extravasation and provides a setting in which the biophysical properties of the infusion solution can be rigorously controlled while allowing for the change in P50. Therefore, experimenting with vesicles that encapsulate Hb formulated with different P50 values provides the unique opportunity to investigate how oxygen affinity regulates oxygen delivery to the tissue by the microcirculation, a value not attainable by lowering RBC Hb P50 by the administration of sodium cyanate, which may introduce changes in tissue metabolism (7). In addition, RBC and HbV are different in size, flow pattern, homogeneous distribution in the plasma phase, and the mechanism of oxygen unloading in capillaries, and direct comparison between RBC and HbV is impossible. All these conditions indicate that the optimal P50 should be different in HbV and RBC.
In the present study, we investigated the microvascular effects of restoring oxygen-carrying capacity in conditions of extreme hemodilution, introducing by exchange transfusion identical amounts of Hb-carrying vesicles in which oxygen affinity was specifically controlled so that P50 was either 8 or 29 mmHg. The P50 value of 8 mmHg was chosen because it is similar to that of a recently developed oxygen carrier that is effective at a low concentration (2–4, 22). In these experiments, the hemodilution protocols were performed using a recombinant albumin solution (13) as the plasma expander.
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METHODS |
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Preparation of HbV with different P50. HbV were prepared under sterile conditions as previously reported (12, 15). Hb was purified from outdated donated blood provided by the Hokkaido Red Cross Blood Center (Sapporo, Japan) and the Japanese Red Cross Society (Tokyo, Japan). The encapsulated purified Hb (38 g/dl) contained 0 or 14.7 mM PLP (Sigma Chemical, St. Louis, MO) as an allosteric effector at a molar ratio of [PLP]/[Hb] = 0 or 2.5, respectively. The lipid bilayer was composed of a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, cholesterol, and 1,5-bis-O-hexadecyl-N-succinyl-L-glutamate at a molar ratio of 5:5:1 (Nippon Fine Chemical, Osaka, Japan) and 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-poly(ethylene glycol) (0.3 mol% of the total lipid; NOF, Tokyo, Japan) (17). HbV with a 250-nm diameter were suspended in a physiological saline solution in which [Hb] = 10 g/dl, sterilized with filters (Dismic, pore size 0.45 µm; Toyo Roshi, Tokyo, Japan), and deoxygenated with N2 bubbling for storage (14). The content of lipopolysaccharide was <0.1 EU/ml.
Before use, the HbV suspension ([Hb] = 10 g/dl, 8.6 ml) was mixed with a solution of recombinant human serum albumin (rHSA 25%, 1.4 ml; Nipro, Osaka, Japan) to regulate the rHSA concentration in the suspending medium of the vesicles to 5 g/dl. Under this condition, the colloid osmotic pressure of the suspension is 20 mmHg (Wescor 4420 colloid osmometer; Wescor, Logan, UT) (12). As a result, the Hb concentration of the suspension was 8.6 g/dl.
In a previous study (16), HbV were suspended in 8 g/dl HSA. However, we changed to 5 g/dl rHSA because it showed better microvascular perfusion in the hamster window model (i.e., increased red cell velocity and functional capillary density) than 8 g/dl HSA. The suspension was filtered through sterile filters (pore size 0.45 µm; Millipore, Billerica, MA). The characteristics of HbV are listed in Table 1, with all parameters being almost identical except oxygen affinity (HbV8, P50 = 8 mmHg; HbV29, P50 = 29 mmHg).
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After level 2, the animals were randomly divided into three experimental groups by being assigned to an experimental group according to a sorting scheme based on a list of random numbers (1). Level 2 exchange was followed by level 3 exchange. Hemodilution with 5% rHSA solution was continued with one group of the level 2 hemodiluted animals, the experimental group rHSA, until Hct was decreased to 11% of baseline (Fig. 2). The test materials were studied by assigning the remainder of the level 2 animals to groups labeled HbV8 (P50 = 8 mmHg) and HbV29 (P50 = 29 mmHg) and were hemodiluted using these materials, reducing Hct to 11%. Plasma Hb concentrations derived for HbV8 and HbV29 after exchange of 35% blood volume are estimated around 2.0–2.3 g/dl for both groups (35% of estimated total Hb content) (21).
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Blood chemistry and biophysical properties. Arterial blood was collected in heparinized glass capillaries (0.05 ml) and immediately analyzed for arterial Po2 (PaO2), arterial Pco2 (PaCO2), base excess (BE), and pH (Blood Chemistry Analyzer 248; Bayer, Norwood, MA). The comparatively low PaO2 and high PaCO2 values of these animals is a consequence of their adaptation to a fossorial environment. Blood samples for viscosity and colloid osmotic pressure 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+) with a cone spindle (CPE-40; both from Brookfield Engineering Laboratories, Middleboro, MA) at a shear rate of 160 s–1. Colloid osmotic pressure (COP) was measured using the Wescor 4420 colloid osmometer (23).
Functional capillary density. Functional capillary density (FCD; in cm–1) is the total length of RBC-perfused capillaries divided by the area of the microscopic field of view (21). Capillary segments were considered functional if RBC were observed to transit over a 30-s period. FCD was tabulated from the capillary lengths with RBC flow in an area comprising 10 successive microscopic fields (420 x 320 µm). Detailed mappings were made of the chamber vasculature to study the same microvessels throughout the experiment.
Microhemodynamic parameters.
Arteriolar and venular blood flow velocities were measured online using the photodiode cross-correlation technique (8) (Fiber Optic Photo Diode and Velocity Tracker Correlator model 102B; Vista Electronics, Ramona, CA). The centerline velocity (V) was corrected according to vessel size to obtain the mean RBC velocity (11). The video image shearing technique was used to measure vessel diameter (D) online. Blood flow was calculated from the measured parameters as (Q) = V
(D/2)2.
Microvascular PO2 distribution. High-resolution microvascular PO2 measurements were made using phosphorescence-quenching microscopy (18), a method based on the oxygen-dependent quenching of phosphorescence emitted by albumin-bound metalloporphyrin complex after pulsed light excitation. Phosphorescence microscopy is not dependent on the level of dye within the tissue, and the decay time is inversely proportional to the PO2 level. The phosphorescence decay curves were converted to oxygen tensions by using a fluorescence decay curve fitter (model 802; Vista Electronics) (9). This technique has been used in this animal preparation and others for both intravascular and extravascular oxygen tension measurements, because albumin exchange between plasma and tissue allows for sufficient concentrations of albumin-bound dye within the interstitium to achieve an adequate signal-to-noise ratio. Animals received a slow intravenous injection of 15 mg/kg body wt at a concentration of 10.1 mg/ml of a palladium-meso-tetra(4-carboxyphenyl)porphyrin (Porphyrin Products, Logan, UT). PO2 measurements were made 20 min after porphyrin injection, allowing it to be distributed to all the tissues.
In our system, intravascular measurements are made by placing an optical rectangular window (5 x 40 µm) within the vessel of interest, with the longest side of the rectangular slit positioned parallel to the vessel wall. Tissue PO2 is measured in regions void of large vessels within intercapillary spaces with an optical window size of 10 x 10 µm, which allows us to precisely establish the localization of the PO2 measurements in arterioles, venules, and the interstitium (20). The phosphorescence decay due to quenching at a specific PO2 yields a single decay constant, and in vitro calibration has been demonstrated to be valid for in vivo measurements. Intravascular and perivascular PO2 measurements were made in the arterioles studied, and intravascular PO2 measurements were made in venules. Interstitial tissue PO2 was measured in regions distant from visible underlying and adjacent vessels.
Tissue oxygen delivery and extraction.
The microvascular methodology used in our studies allows a detailed analysis of oxygen supply in the tissue. Calculations of O2 delivery, defined as the amount of oxygen delivered by the arterioles to the microcirculation per unit time normalized relative to baseline, and O2 extraction, defined as the amount of oxygen released by blood to the tissue by the microcirculation per unit time normalized relative to baseline, were made using Eqs. 1 and 2:
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 | (2) |
is the oxygen-carrying capacity of Hb at 100% saturation (or 1.34 ml O2/g Hb), Sa% indicates the arteriolar oxygen saturation of RBC or HbV, S(a-v)% indicates the arteriovenous difference in oxygen saturation of RBC or HbV, (1 – Hct) is the fractional plasma volume (and converts the equation from units per dl of plasma to per dl of blood), 
is the solubility of oxygen in plasma and is equal to 3.14 x 10–3 ml O2/dl plasma mmHg, PaO2 is the arteriolar partial pressure of oxygen, P(a-v)O2 is the arteriovenous difference in PO2, and Q is the microvascular flow for each microvessel as a percentage of baseline. The oxygen dissociation curves were determined as described before. In this analysis, microvascular Hct was corrected according to the findings of Lipowsky and Firrell (10). Experimental procedure. Baseline systemic, microvascular, and hemodynamic characterizations were performed before the start of the exchange. After each exchange and a stabilization period of 10 min, systemic and/or microvascular measurements were performed. Exchanges began every hour. After the level 3 exchange transfusion, the same measurements were repeated, and then the PO2 distribution was determined using phosphorescence-quenching microscopy (9). The duration of the experiment was 3–4 h.
Data analysis. Results are presented as means ± SD unless otherwise noted. All 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 higher or lower than baseline. The same vessels and functional capillary fields were followed so that direct comparisons to their baseline levels could be performed, allowing for more robust statistics for small sample populations. For repeated measurements, time-related changes were assessed by analysis of variance (ANOVA). Data within each group were analyzed using ANOVA for nonparametric repeated measurement, and when appropriate, post hoc analyses were performed with the Dunn's multiple comparison tests. For level 3 exchange, groups were analyzed using one-way ANOVA, and post hoc analyses were performed with the Bonferroni post tests. All statistics were calculated using GraphPad Prism 4.01 (GraphPad Software, San Diego, CA). Changes were considered statistically significant if P < 0.05.
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RESULTS |
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Twenty-four animals (55–65 g body wt) entered into the exchange-transfusion (hemodilution) protocol, and all tolerated the experiment without any visible discomfort. Microvascular studies were completed in six preparations for each test material, namely, the level 2 rHSA, HbV8, and HbV29. The data were analyzed using a model for computing oxygen delivery to the tissue at the microscopic level.
Hematological changes.
The exchange-transfusion protocol resulted in a final Hct ranging from 11.0 ± 0.5 to 11.4 ± 0.6%. The HbV8 and HbV29 groups had a final plasma Hb concentration of 2.1 ± 0.1 g/dl, which increased the total Hb concentration in blood (RBC + Hb in plasma) to 5.7 ± 0.2–0.3 g/dl after completion of the level 3 exchange transfusion. Thus oxygen-carrying capacities at this level were similar to those found at level 2, where total blood Hb concentration was 5.7 ± 0.3 g/dl (Hct 18.1 ± 0.7) (Table 2).
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Changes in the systemic parameters are presented in Fig. 3. Mean arterial pressure was statistically lower for the extreme hemodilution tests with rHSA and the HbV29 group and attained the highest value with HbV8 viscosity. Heart rate after hemodilution followed by exchange transfusion with the HbV solutions was 10% higher than baseline at the level 3 exchange. The slight increase in heart rate was not statistically different.
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Colligative properties.
Blood viscosities and COP after level 3 exchange were sampled at 1 h and 10 min after completion of the exchange. Table 3 shows that blood viscosity ranges from 1.6 cp (plasma 0.9 cp) for rHSA to 2.0 cP (plasma 1.0 cp) for the HbV groups.
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Microhemodynamics.
After level 3 exchange, arteriolar and venular diameters were not statistically different from baseline for any of the groups. Arteriolar flow velocities attained the highest value for the HbV8 group, being 1.90 relative to baseline, which was statistically significant. The same effect was found in the venular microcirculation, where blood flow velocity was 2.20 relative to baseline. HbV29 exchange transfusion lowered both arteriolar and venular velocities relative to the values attained at the level 2 exchange. However, venular velocity in this group was statistically significantly higher than in baseline. Notably, the level 2 hemodilution with rHSA caused significantly higher blood flow velocities in the arteriolar and venular microcirculation (Fig. 4).
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Functional capillary density.
The number of capillaries with RBC passage upon level 3 hemodilution in the rHSA, HbV8, and HbV29 groups was 62 ± 9, 76 ± 12, and 72 ± 13% of baseline, respectively. These values were statistically different from baseline but not statistically different with respect to each other (Fig. 6).
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Oxygen tension measured using phosphorescence microscopy after level 3 exchange transfusion in the rHSA, HbV8, and HbV29 groups showed that these materials produced virtually identical distributions of arteriolar microvascular PO2 (arterioles averaged 49.5 mmHg), although HbV8 tended to be higher (Fig. 7). The decrease of RBC from level 2 to level 3 did not decrease the arteriolar PO2. Venular PO2 after level 3 was significantly lower than at level 2 exchange in all cases (rHSA, 7.2 ± 3.2 mmHg; HbV8, 15.1 ± 3.7 mmHg; HbV29, 9.6 ± 4.2 mmHg).
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Oxygen delivery and extraction.
Figure 8 shows the results of the analysis for delivery and release of oxygen by the microcirculation. It is apparent that exchanging RBC for HbV8 maintains oxygen delivery to the tissue, whereas HbV29 reduces this by 20%, and continued hemodilution with a non-oxygen-carrying material significantly depresses oxygen delivery to the tissue, reducing this to half of that attained at the level 2 hemodilution.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In the hemodilution procedures of this study, blood was exchanged with a rHSA solution as a colloidal plasma expander, which was the same suspending medium used for the Hb vesicles. Therefore, in these experiments, we can make a direct comparison between an oxygen-carrying and non-oxygen-carrying blood substitute, uncomplicated by the presence of additional materials. Our results show that the level 2 hemodilution with rHSA leads to maintained functional capillary density and significantly improved arteriolar and venular blood flow, although somewhat lowered central blood pressure. The latter finding is not necessarily negative and may reflect a lowered overall peripheral vascular resistance due to the decrease of blood viscosity after hemodilution. The fact that microvascular flow is significantly increased indicates that the level 2 hemodilution with rHSA provides the tissue with adequate microvascular perfusion and that this colloid is an adequate plasma expander.
Average oxygen delivery and extraction were somewhat greater for HbV8 than for HbV29. These are calculated values and are not statistically significantly different; however, the same difference was found in all micro and macro parameters measured in this study.
The level 2 hemodilution and the succeeding level 3 hemodilution with either HbV8 and HbV29 resulted in the same total Hb concentration in the circulation (5.7 and 5.8 g Hb/dl); however, oxygen delivery was lower with HbV29 and lowest with rHSA, as might be expected due to the low Hb content (3.7 g Hb/dl) in the absence of plasma Hb for the rHSA group. Therefore, because all groups had the same Hct at the level 3 hemodilution, the sustained oxygen consumption and tissue PO2 relative to the rHSA group clearly demonstrate that Hb vesicles release oxygen. However, the vesicles with the lowest P50 provide an oxygen delivery capacity identical to that of blood at level 2 hemodilution, whereas vesicles with a high P50 lower oxygen delivery at the microcirculatory level, an effect probably caused by the decreased blood flow associated with HbV29.
The differences in tissue PO2, mean arterial blood pressure, and arteriolar blood flow between HbV8 and HbV29 show that in designing a blood substitute, it is not sufficient to provide adequate oxygen-carrying capacity. Once a suitable oxygen carrier is available, it also must be able to maintain or enhance other circulatory transport parameters, particularly flow. The Hb vesicles used in this study are vasoinactive, and the difference in P50 appears to be a factor in improving flow condition that is not related to vasoactivity. An explanation for this may be related to the inherent variability of tissue PO2 shown in this and other studies (4, 22), which may be enhanced in extreme hemodilution. This variability determines that if average tissue PO2 is low, portions of the tissue may become anoxic. Introducing a small quantity of a low-P50 Hb oxygen carrier into the circulation will deliver oxygen only to those parts of the tissue where the anoxic threshold is passed, thus eliminating the inherent variability of oxygen delivery shown by the variability of tissue PO2.
Considering the significantly improved blood pressure and the trend toward higher flow for HbV8 (in the absence of vasoconstriction and changes in the rheological properties of blood), it is possible that in conditions of extreme hemodilution the cardiac function should be improved because of the proposed more homogenous heart tissue oxygenation using HbV8 vs. HbV29.
In summary, the present results show that either HbV8 or HbV29 are efficient oxygen carriers that do not cause vasoactivity. The experiments were carried out using rHSA as a hemodiluent, and this material was adequate as a plasma volume substitute. Oxygen extraction was similar for both oxygen carriers; however, HbV8 appeared to be beneficial at the systemic level, because base excess remained at baseline levels, whereas it was decreased for HbV29. This finding suggests that improved tissue PO2 and microcirculatory oxygen delivery may be efficient in other tissues. The improvement obtained may be specific to the conditions of these experiments in which the vesicles were tested for their capacity to restore tissue PO2, FCD, and oxygen extraction in the microcirculation during extreme hemodilution. The significant differences in the tissue oxygen parameters produced by the presence of low-P50 Hbs vs. an identical oxygen carrier with normal P50 suggests that small amounts of Hbs with high oxygen affinity may have therapeutic effects in the treatment of ischemic conditions (6).
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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) in hamster window chamber. Am J Physiol Heart Circ Physiol 287: H1609–H1617, 2004.This article has been cited by other articles:
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  P. Cabrales, A. G. Tsai, R. M. Winslow, and M. Intaglietta Extreme hemodilution with PEG-hemoglobin vs. PEG-albumin Am J Physiol Heart Circ Physiol, December 1, 2005; 289(6): H2392 - H2400. [Abstract] [Full Text] [PDF]
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H. Sakai, P. Cabrales, A. G. Tsai, E. Tsuchida, and M. Intaglietta Oxygen release from low and normal P50 Hb vesicles in transiently occluded arterioles of the hamster window model Am J Physiol Heart Circ Physiol, June 1, 2005; 288(6): H2897 - H2903. [Abstract] [Full Text] [PDF]
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P < 0.05 relative to baseline; 
P < 0.05 relative to level 3 rHSA; ¶P < 0.05, level 3 with HbV8 vs. level 3 with HbV29.
P < 0.05 compared with level 2 with rHSA; 