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Am J Physiol Heart Circ Physiol 289: H2392-H2400, 2005. First published July 15, 2005; doi:10.1152/ajpheart.00225.2005
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Extreme hemodilution with PEG-hemoglobin vs. PEG-albumin

Pedro Cabrales,1 Amy G. Tsai,1,2 Robert M. Winslow,2,3 and Marcos Intaglietta1,2

1La Jolla Bioengineering Institute, La Jolla; 3Sangart, Incorporated, San Diego; and 2Department of Bioengineering, University of California, San Diego, La Jolla, California

Submitted 8 March 2005 ; accepted in final form 11 July 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Isovolemic hemodilution to 11% systemic hematocrit was performed in the hamster window chamber model using 6% dextran 70 kDa (Dx 70) and 5% human serum albumin (HSA). Systemic and microvascular effects of these solutions were compared with polyethylene glycol (PEG)-conjugated 5% albumin (MPA) and PEG-conjugated 4.2% Hb (MP4). These studies were performed for the purpose of comparing systemic and microvascular responses of PEG vs. non-PEG plasma expanders and similar oxygen-carrying vs. noncarrying blood replacement fluids. Mean arterial blood pressure was statistically significantly reduced for all groups compared with baseline (P < 0.05), HSA, MPA, and MP4 higher than Dx 70 (P < 0.05). MP4 and MPA had a significantly higher cardiac index than HSA and Dx 70, in addition to a positive base excess. Microvascular blood flow and capillary perfusion were significantly higher for the PEG compounds compared with HSA and Dx 70. Intravascular PO2 for MP4 and MPA was higher in arterioles (P < 0.05) compared with HSA and Dx 70, but there was no difference in either tissue or venular PO2 between groups. Total Hb in the MP4 group was 4.8 ± 0.4 g/dl, whereas the remaining groups had a range of 3.6–3.8 g/dl. The hemodilution results showed that PEG compounds maintained microvascular conditions with lower concentrations than conventional plasma expanders. Furthermore, microvascular oxygen delivery and extraction in the window chamber tissue were significantly higher for the PEG compounds. MP4 was significantly higher than MPA (P < 0.05) and was not statistically different from baseline, an effect due to the additional oxygen release to the tissue by the Hb MP4.

microcirculation; functional capillary density; cardiac output; oxygen release; blood substitutes; plasma expanders; polyethylene glycol-albumin; polyethylene glycol-hemoglobin

RECENT DEVELOPMENTS IN THE field of blood substitutes based on the modification of human Hb (Hb) have led to the formulation of polyethylene glycol (PEG)-conjugated Hb compounds (36). They have a high oxygen affinity, moderated viscosity, and high colloidal osmotic pressure (COP). PEG-conjugated Hb (PEG-Hb) sustains systemic and microvascular function in extreme hemodilution [hematocrit (Hct) 11%] at a plasma concentration of ~1.0 g/dl and total blood Hb [plasma + red blood cells (RBCs)] concentration of 4.5–5.0 g/dl (36).

PEG-Hb was shown to be free of hypertension in a rat model (37) and was also shown to release oxygen in an artificial capillary in a manner virtually identical to native RBCs (22). On the basis of these observations, a new PEG-Hb was designed [MP4 (PEG-conjugated human Hb); Sangart, San Diego, CA] with the potential for large-scale production as a blood substitute (3, 33).

Tsai et al. (32) and Cabrales et al. (12) found that PEG-Hb maintained microvascular function and functional capillary density (FCD) within normal physiological limits after extreme hemodilution. Similar results were obtained after extreme hemodilution using the high-viscosity plasma expander dextran 500 kDa, which maintained FCD by increasing plasma viscosity to 2.2 cP and blood viscosity to 2.8 cP. PEG-Hb compounds in the dosages normally used do not yield to increase plasma or blood viscosity. Therefore, PEG-Hb efficacy should be attributable to either the modest increase in oxygen-carrying capacity (1 g Hb/dl), their high oxygen affinity, or the increased efficiency in combination with RBCs releasing oxygen more efficient to the tissues (15, 32).

PEG conjugation with albumin yields a molecule [PEG-conjugated 5% albumin (MPA)] physically similar to PEG-Hb (MP4), with the difference being its non-oxygen-carrying capacity. MPA allows us to test the difference between the oxygen-carrying and noncarrying capacity of two virtually identical molecules. Winslow et al. (37) compared MPA and MP4 in a hemodilution rat model where the animals were 100% exchanged with these materials, showing MP4 can support life at zero Hct. Consequently, MP4 presented additional properties than plasma expansion due to PEG-Hb oxygen-carrying capacity, which should become evident by the direct analysis of its effects at the level of the microcirculation.

Extreme isovolemic hemodilution is an experimental model where systemic Hct is lowered to the point of minimal intrinsic oxygen-carrying capacity. This critical point is a short edge, where changes in oxygen-carrying capacity and other blood properties of the test material can either sustain, improve, or deteriorate systemic and microvascular parameters. The extreme hemodilution model is well suited to analyze the properties of plasma expander and blood substitutes. The acute anemic conditions magnify the effects of the compounds. At this condition, the system is depressed and not fully capable of compensating for the changes in blood composition.

The principal aim of this study was to compare the effects of the increase in oxygen-carrying capacity due to the presence of Hb in MP4 with those that may be attributable to a PEG protein with more or less identical properties. In principle, MPA is rendered null and void of any pharmacological activity, since neither PEG, albumin, nor their combination had reported vasoactive properties. To accomplish this goal, we compared systemic [including measurement of cardiac output (CO)] and microvascular responses in extreme hemodilution attained via a two-step procedure with 70-kDa dextran (Dx 70) with a Hct of 18% followed by an additional hemodilution to Hct 11% using MPA (5 g/dl; Sangart) and MP4 (4.2 g/dl, PEG-Hb, P50 = 5.4 mmHg; Sangart). Both molecules were modified using the maleamide PEG process (33). We also used 5% human serum albumin (HSA) to differentiate the effects due to albumin per se and MPA and compared this with previous findings with 6% Dx 70, conventional plasma expanders with similar molecular weight.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 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 chamber window. Animal handling and care followed the NIH Guide for the Care and Use of Laboratory Animals. Experimental protocol was approved by the local animal care committee. The hamster chamber window model is widely used for microvascular studies in the unanesthetized state, and the complete surgical technique is described in detail elsewhere (13, 14). Briefly, the animal was prepared for chamber implantation with a 50 mg/kg ip injection of pentobarbital sodium anesthesia. After hair removal, sutures were used to lift the dorsal skin away from the animal, and one frame of the chamber was positioned on the animal’s back. The chamber consists of two identical titanium frames with a 15-mm circular window (12-mm-diameter circular visible field). With the aid of backlighting and a stereomicroscope, one side of the skinfold was removed following the outline of the window until only a thin layer of retractor muscle and the intact subcutaneous skin of the opposing side remained. The cover glass was placed on the exposed skin held in place by the other frame of the chamber. The intact skin of the other side was exposed to the ambient environment. The animal was allowed at least 2 days for recovery before the preparation was assessed under the microscope for any signs of edema, bleeding, or unusual neovascularization. Barring these complications, the animal was anesthetized again with pentobarbital sodium. Arterial and venous catheters were implanted in the carotid artery (PE-50) and jugular vein (PE-50), respectively, and filled with a heparinized saline solution (30 IU/ml) to ensure patency at the time of experiment. Catheters were tunneled under the skin and exteriorized at the dorsal side of the neck where they were attached to the chamber frame with tape. After the initial surgery (3–4 days), the microvasculature was examined, and only animals passing an established systemic and microcirculatory inclusion criteria (which includes having tissue void of low perfusion, inflammation, and edema) were entered into the study (31).

Inclusion criteria. Animals were suitable for the experiments if 1) systemic parameters were within normal range, namely, heart rate (HR) >340 beats/min, mean arterial pressure (MAP) >80 mmHg, systemic Hct >45%, and arterial oxygen partial pressure (PaO2) >50 mmHg; and 2) microscopic examination of the tissue in the chamber observed under x650 magnification did not reveal signs of edema or bleeding. Body weight was monitored before and the next day after surgeries, and, at the time of the experiment, significant reductions (>4 g) in body weight prevented the use of animal compromise during animal instrumentation. All the animals included in the study were observed behaving normal before the inclusion in the experimental protocol.

Test materials. MP4 a Hb solution was prepared and characterized as described previously (33). Briefly, outdated human packed RBCs, obtained from the San Diego Blood Center, were washed with saline, lysed with distilled water, and dialyzed to achieve an Hb concentration of 4.2 g/dl in lactated Ringer solution. Maleimide-activated PEG-Hb (Mal-PEG-Hb) was produced by the reaction of human Hb with 2-iminothiolane to introduce sulfhydryl groups at surface {epsilon}-amino groups (lysine), which were then conjugated to six strands per tetramer of Mal-PEG [mol mass 5 kDa (NOF, Tokyo, Japan), see Ref. 33]. Preparation of Mal-PEG-albumin was done by the same procedure, substituting Hb for albumin. Size-exclusion chromatography was performed using an AKTA Purifier-10 instrument (Pharmacia) using two columns in series. Samples were eluted using PBS at pH 7.4 and a flow rate of 0.5 ml/min at room temperature. Albumin (5% wt/vol) was prepared by adding 5 ml of 25% HSA (12.5 g albumin; Baxter Healthcare, Toronto, Canada) to 20 ml of 0.9% sodium chloride, USP, to obtain 25 ml 5% albumin. Dx 70 (Pharmacia), 6% wt/vol in 0.9% saline, was used as a colloid control solution.

The test solutions (Table 1) were as follows: 1) Dx 70, containing 6 g/dl Dx 70; 2) HSA, 5 g/dl HSA; 3) MPA, containing 5.0 g/dl Mal-PEG-albumin in lactated Ringer, USP; and 4) MP4, containing 4.2 g/dl Mal-PEG-Hb in lactated Ringer, USP. The concentration of MPA was chosen to match COP and viscosity with MP4 (Table 1).


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  		Table 1. Physical characteristics of the solutions

 
Acute isovolemic exchange transfusion (hemodilution) protocol. Progressive hemodilution to a final systemic Hct level of 11% was accomplished with three isovolemic exchange steps. This protocol is described in detail in our previous reports (6–9, 11, 27, 32). Briefly, the volume of each exchange-transfusion step was calculated as a percentage of the blood volume, estimated as 7% of body weight. An acute anemic state was induced by lowering systemic Hct to 18% with two steps of progressive isovolemic hemodilution using 6% Dx 70, exchange levels 1 and 2. Level 1 exchange was 40% of blood volume, and level 2 was 35% of blood volume.

After the level 2 exchange, animals were randomly divided into four experimental groups by assigning each animal 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, exchanging 35% of blood volume using Dx 70 (group 1), HAS (group 2), MPA (group 3), or MP4 (group 4) to a Hct of 11%.

Because mixed blood and dilution material were withdrawn during the exchanges, a 110% blood volume exchange was needed to reduce the Hct to 11% (6–9, 11, 27, 32). The transfusion solutions were filtered (in line, 0.22 µm filter, at a rate of 100 µl/min, "33" syringe pump; Harvard Apparatus, Holliston, MA) and infused in the jugular vein catheter. Blood was simultaneously withdrawn at the same rate from the carotid artery catheter according to a previously established protocol (6–9, 11, 27, 32). Blood samples were withdrawn from level 3 exchange animals at the end of the experiment for subsequent analysis of viscosity and colloid osmotic pressure. The duration of the experiments was 4 h to complete the degree of hemodilution. Each exchange and the respective observation time point postexchange were fully completed in 1 h. Systemic and microcirculation data were taken after a 5-min stabilization period.

Experimental setup. The unanesthetized animal was placed in a tube that restricted movement but did not impede respiration and was given for 30 min to allow adjustment before control systemic parameters (MAP, HR, blood gases, and Hct) were measured. The animal was then fixed to the stage of a transillumination intravital microscope (BX51WI; Olympus, New Hyde Park, NY). The tissue image was projected on a charge-coupled device camera (COHU 4815) connected to a videocassette recorder (AG-7355; JVC, Tokyo, Japan) and viewed on a monitor. Measurements were made using a x40 (LUMPFL-WIR, numeric aperture 0.8; Olympus) water-immersion objective. Contrast between RBCs and tissue was enhanced with a BG12 (420 nm) bandpass filter.

Fields of observations and vessels were chosen for study at locations in the tissue where vessels were in sharp focus. The same fields and vessels were used throughout the experiment, allowing for direct comparisons with baseline levels.

Blood chemistry and biophysical properties. Arterial blood was collected in heparinized glass capillaries (0.05 ml) and immediately analyzed for PaO2, PaCO2, base excess (BE), and pH (Blood Chemistry Analyzer 248; Bayer, Norwood, MA). The comparatively low PaO2 and high PaCO2 of these animals is a consequence of their adaptation to a fossorial environment. Viscosity was measured in a DV-II plus (Brookfield Engineering Laboratories, Middleboro, MA) cone/plate viscometer with a CPE-40 cone spindle at a shear rate of 160/s. Colloid osmotic pressure was measured using a 4420 Colloid Osmometer (Wescor, Logan, UT; see Ref. 35).

Hb oxygen saturation. Oxygen saturation of Hb was investigated by deoxygenation of oxygen-equilibrated oxy-Hb in the Hemox buffer (pH 7.4) at 37.6°C using a Hemox Analyzer (TCS Scientific, New Hope, PA). The analyzer measures the O2 pressure with a Clark-type O2 electrode (Yellow Springs Instrument, Yellow Springs, OH) and simultaneously calculates the Hb saturation via a dual-wavelength spectrophotometer. The oxygen equilibrium curve (OEC) for MP4 was measured using the method described previously (34). OEC for hamster RBCs was determined from freshly collected blood.

CO measurement. CO was measured by a modified thermodilution technique (5) in a different group of animals not used for microvascular studies due to the complexity of the setup and the difficulty of positioning the instrumented animal on the microscope. These animals were characterized in terms of systemic parameters to ensure that they presented the same characteristics as those used in the microcirculatory studies. Animals assessed for CO were randomly assigned to different experimental groups. CO was measured 15–20 min after exchange.

FCD. FCD is the total length of RBC perfused capillaries divided by the area of the microscopic field of view (31). Capillary segments are considered functional if RBCs are observed to transit over a 30-s period. FCD was tabulated from the capillary lengths with RBC flow in an area comprised of 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 velocity was measured on-line using the photodiode cross-correlation technique (Fiber Optic Photo Diode and Velocity Tracker model 102B; Vista Electronics, San Diego, CA; see Ref. 17). The centerline velocity (V) was corrected according to vessel size to obtain the mean RBC velocity (21). The video image shearing technique was used to measure vessel diameter (D) on-line (16). Blood flow was calculated from the measured parameters as Q = {pi}V x (D/2)2. This calculation assumes a parabolic velocity profile and has been found to be applicable to tubes of 15–80 µm internal diameter and for Hct values in the range of 6–60% (21). Wall shear stress (WSS) was defined by WSS = WSR x {eta}, where WSR is the wall shear rate given by 8VD–1, and {eta} is microvascular blood viscosity or plasma viscosity.

In conditions of extreme hemodilution with Hct ~11%, the contribution of RBCs to the total viscosity of blood is linear and amounts to ~0.70 cP, which is the difference between blood and plasma viscosity. According to Lipowsky and Firrell (19), the ratio between arteriolar-venular and systemic Hct in extreme hemodilution converges to 0.7 for Hct ~10%. Therefore, we corrected our extreme hemodilution viscosity data by linearly reducing the viscosity RBC contribution by 70%. The same procedure was used for the normal blood data, where the Hct reduction was 0.58 for arterioles and 0.68 for venules (19, 20). However, because at normal Hct, blood viscosity was not linearly proportional to Hct, we used actual viscosity vs. Hct (dilution with hamster blood) data to obtain the corrected value for blood viscosity (9, 11).

Microvascular PO2 distribution. High-resolution noninvasive microvascular PO2 measurements were made using phosphorescence quenching microscopy (PQM; see Ref. 26). PQM is based on the oxygen-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 the circulation in the interstitial tissue (4, 6, 8, 10, 27, 30). Interstitial PO2 measurements have been found to be identical to simultaneous measurements made with recessed oxygen electrodes (4). Tissue PO2 was measured in tissue regions in between functional capillaries. The reported values are the average of several determinations in several animals. PQM allows for precise localization of the PO2 measurements without subjecting the tissue to injury. These measurements provide a detailed understanding of microvascular oxygen distribution and show whether oxygen is delivered to the interstitial areas.

The system setup has been described in previous references (18, 25). Animals received a slow intravenous injection of 15 mg/kg body wt at a concentration of 10.1 mg/ml palladium-meso-tetra(4-carboxyphenyl)porphyrin (Porphyrin Products), which is allowed to circulate for 20 min before PO2 measurements. The phosphorescence was excited by pulsed light (30 Hz, 4 µs duration) for a period of <5 s, and the measurement site was microscopically vignetted by an adjustable slit. For intravascular measurements, an optical rectangular slit ~5 x 35 µm was positioned longitudinally within the vessel of interest. For interstitial tissue measurements, a 15 x 10 µm slit was placed in intercapillary spaces in regions void of large vessels. The phosphorescence decay curves were analyzed off-line, using a standard single exponential least-squares numerical fitting technique, and the resultant time constants were applied to the Stern-Volmer equation to calculate PO2 using predetermined parameters corrected for this animal model (18).

Oxygen delivery and extraction. The microvascular methodology used in our studies allows a detailed analysis of oxygen supply to the tissue. Calculations of oxygen delivery and extraction are based on a mass balance analysis of the rate of oxygen entrance and exit in the microcirculation determined in arterioles and venules: O2 delivery is defined as the amount of oxygen per unit time delivered by the arterioles to the microcirculation normalized relative to control, and extraction (O2 extraction) is defined as the amount of oxygen released by blood in the microcirculation per unit time normalized relative to control (7, 10, 24):


{H0022505pH2392eq1}

(1)


{H0022505pH2392eq2}

(2)
where RBCHb is the Hb in RBCs expressed in grams per deciliter of blood, PlasmaHb is the cell free Hb in grams per deciliter of blood, {gamma} is the oxygen carrying capacity of Hb at 100% saturation or 1.34 ml O2/g Hb, SA% is the arteriolar oxygen saturation of RBCs, SA% is the arteriolar oxygen saturation of MP4 and the subscript A-V indicates the difference between arterioles and venules, 1 – Hct is the fractional plasma volume and converts the equation from per deciliter of plasma to per deciliter of blood, {alpha} is the solubility of oxygen in plasma equal to 3.14 x 10–3 ml O2/dl plasma mmHg, PaO2 is the arteriolar partial pressure of oxygen, PO2A-V is the arteriolar/venular difference in PO2, and Q is the microvascular flow for each microvessel as percent of baseline. The OECs were determined using the Hemox Analyzer (TCS). In this analysis, microvascular Hct was corrected according to the findings of Lipowsky and Firrell (19). This correction is minimal for the hemodiluted groups and the same for all of them. Therefore, microvascular Hct does not affect intergroup comparisons. Control Hct was also corrected according to the findings of Lipowsky and Zweifach (21) and Baker and Wayland (2). This correction was developed for the cat mesentery and is assumed to be applicable to chamber window tissue, which presents a similar distribution of Hct in the microvascular network (2, 20).

Data analysis. Results are presented as means ± SD unless otherwise denoted. Data within each group were analyzed using one-way ANOVA and, when appropriate, post hoc analyses performed with the Bonferroni’s multiple-comparison tests. 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 and 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. All statistics were calculated using GraphPad Prism 4.01 (GraphPad Software, San Diego, CA). Changes were considered statistically significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Twenty two animals were entered in the microcirculation study. All animals tolerated the entire hemodilution protocol without visible signs of discomfort. The animals were randomly assigned to the following experimental groups: Dx 70 (n = 6); HSA (n = 4); MPA (n = 6); and MP4 (n = 6). CO was studied in a separate group of animals that underwent an identical hemodilution protocol. Five animals were studied in each group. A total of 42 animals was included in the hemodilution protocol for the present study. Systemic data were obtained from groups used for microvascular and CO studies. Similarities between groups used for microcirculation and CO were statistically verified for baseline, level 1, level 2, and level 3 in each group (P > 0.20).

CO. CO and calculated cardiac index (CI; CO/body wt) are presented in Fig. 1. CO increased from baseline 1.4 times after level 1, and, after level 2, CO was 1.6 times baseline. Level 3 exchange with Dx 70 and HSA did not maintain the increased CO, which decreased to 0.83 ± 0.16 and 0.99 ± 0.33 of baseline, respectively. Level 3 exchange with MPA and MP4 maintained the increase in CO, with MPA being 1.44 ± 0.30 and MP4 at 1.34 ± 0.43 of baseline, respectively. The actual values for CO and CI are included in the legend of Fig. 1.



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  		Fig. 1. Cardiac index (CI) after extreme hemodilution for the different plasma expanders. Data are for level 3 exchange: Dx 70, 70 kDa dextran; HSA, human serum albumin; MPA, polyethylene glycol (PEG)-conjugated 5% albumin; MP4, PEG-conjugated 4.2% Hb. Dx 70 hemodilution showed a consistently lower CI relative to baseline, whereas the PEG-Hb caused an increase. P < 0.05 relative to baseline ({dagger}) and compared with level 3 Dx 70 (§). Cardiac output (CO; ml/min) and calculated CI (ml·kg–1·min–1; mean ± SD) in each animal group were as follows: baseline (CO: 17.8 ± 1.6, CI: 218 ± 28, n = 20); level 1 (CO: 25.4 ± 4.1, CI: 288 ± 30); level 2 (CO: 28.5 ± 4.0, CI: 355 ± 35); level 3 Dx 70 (CO: 14.2 ± 1.9, CI: 170 ± 27, n = 5); level 3 HSA (CO: 17.3 ± 1.5, CI: 208 ± 25, n = 5); level 3 MPA (CO: 24.2 ± 1.6, CI: 328 ± 31, n = 5); level 3 MP4 (CO: 23.2 ± 1.8, CI: 315 ± 32, n = 5). n, No. of animals studied.

 
Systemic parameters. The exchange protocol significantly reduced Hct to 28.1 ± 1.7% for level 1 and to 18.0 ± 1.1% for level 2. Hct and Hb for the level 3 exchange are given in Table 2. Exchange using MP4 did not show the same decrease in Hb content compared with the other groups because of the additional Hb contributed by MP4.


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  		Table 2. Macrohemodynamic parameters before and after blood exchange

 
MAP was not changed from baseline (103.6 ± 5.8 mmHg) after level 1 exchange (92.5 ± 10.7 mmHg), and, upon further hemodilution with Dx 70, MAP decreased to 85.9 ± 8.8 mmHg at level 2. Level 3 exchanges lowered MAP, as shown in Table 2. Systemic arterial blood gas analysis showed a statistically significant rise in arterial PO2 from baseline after level 3 for all groups (P < 0.05). Arterial PCO2 and blood pH were not statistically changed from baseline. Blood BE was statistically significantly decreased after level 3 hemodilution compared with baseline in Dx 70 and HSA (Table 2).

Microhemodynamics. The changes in diameter, RBC velocity, and blood flow of large feeding and small arcading arterioles (range 56–85 µm) and small collecting venules and large venular vessels (range 54–96 µm) were measured after each hemodilution step. The arteriolar diameter was unchanged after level 1 exchange. Upon further blood exchange to level 2, arterioles dilated to 1.12 ± 0.24 (n = 74 vessels, P < 0.05) of baseline. This trend reversed after level 3 exchange with Dx 70, HAS, and MP4, resulting in a slight arteriolar vasoconstriction to 0.93 ± 0.25 (Dx 70; n = 18), 0.96 ± 0.22 (HSA; n = 16), and 0.98 ± 0.22 (MP4; n = 20) of baseline. After the level 3 exchange with MPA, arteriolar diameter remained dilated to 1.11 ± 0.26 (MPA; n = 20; P < 0.05 to level 3 Dx 70) of baseline (Fig. 2A).



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  		Fig. 2. Relative changes to baseline in arteriolar and venular hemodynamics at level 1, 2, and 3 hemodilution. Broken line represents baseline level. P < 0.05 relative to baseline ({dagger}) and compared with level 3 Dx 70 (§). Diameters (µm, mean ± SD) in each animal group were as follows: baseline [arterioles (A): 65.1 ± 8.2, n = 74, venules (V): 66.2 ± 10.3, n = 78]; level 1 with Dx 70 (A: 66.4 ± 9.1; V: 62.3 ± 10.6); level 2 with Dx 70 (A: 72.2 ± 10.7; V: 69.2 ± 10.9); level 3 with Dx 70 (A: 62.4 ± 12.5, n = 18, V: 58.2 ± 12.3, n = 16); level 3 with HSA (A: 61.6 ± 12.6, n = 16, V: 60.3 ± 12.1, n = 18); level 3 with MPA (A: 69.2 ± 14.2, n = 20, V: 69.5 ± 14.1, n = 22 ); level 3 with MP4 (A: 63.2 ± 12.8, n = 20, V: 68.2 ± 14.2, n = 22). Red blood cell (RBC) velocities (mm/s, mean ± SD) in each animal group were as follows: baseline (A: 5.1 ± 1.2, V: 1.8 ± 0.6); level 1 with Dx 70 (A: 6.4 ± 1.6; V: 1.9 ± 0.9); level 2 with Dx 70 (A: 7.4 ± 1.6; V: 2.5 ± 1.1); level 3 with Dx 70 (A: 3.4 ± 1.8, n = 18; V: 0.9 ± 1.0, n = 16); level 3 HSA (A: 3.5 ± 2.0, n = 16; V: 0.9 ± 1.1, n = 18); level 3 with MPA (A: 4.1 ± 1.9, n = 20; V: 1.4 ± 1.1, n = 22); level 3 with MP4 (A: 3.9 ± 2.1, n = 20, V: 1.7 ± 1.2, n = 20). Dx 70, 70 kDa dextran.

 
Venular changes resulting from the hemodilution protocol are shown in Fig. 2B as a function of the material infused during the level 3 exchange. Venules responded to level 1 exchange by constricting to 0.94 ± 0.12 of baseline (n = 78, P < 0.05) and returned to baseline after level 2 exchange (1.07 ± 0.14, n = 78). When the exchange protocol was continued to level 3 using Dx 70 and HSA, venules constricted to 0.85 ± 0.16 (Dx 70, n = 16, P < 0.05) and 0.90 ± 0.20 (HSA, n = 18, P < 0.05) of baseline. Level 3 exchange with MPA and MP4 did not change venular diameters from baseline levels, which were 1.09 ± 0.21 (MPA; n = 22; P < 0.05 to level 3 Dx 70) and 1.12 ± 0.15 (MP4; n = 22; P < 0.05 to level 3 Dx 70), respectively.

Figure 2, C and D, shows the change in RBC velocity in arterioles and venules after level 3 exchange for the different groups. Both arteriolar and venular RBC velocity increased after level 1 exchange, rising to 1.42 ± 0.37 (P < 0.05) and 1.36 ± 0.42 (P < 0.05) of baseline, respectively. After level 2 exchange, arteriolar RBC velocity remained increased from baseline (1.45 ± 0.46, P < 0.05), whereas venular RBC velocity returned to baseline levels (1.14 ± 0.29). Level 3 exchange with Dx 70, HSA, MPA, and MP4 reduced arteriolar flow velocity to 0.71 ± 0.41 (Dx 70, P < 0.05), 0.73 ± 0.46 (HSA, P < 0.05), 0.85 ± 0.46 (MPA, P < 0.05), and 0.80 ± 0.38 (MP4) of baseline. Venular RBC velocity decreased statistically significantly after level 3 exchange with Dx 70 and HSA to 0.58 ± 0.49 (Dx 70, P < 0.05) and 0.63 ± 0.41 (HSA, P < 0.05) of baseline. Level 3 exchange using MPA and MP4 did not change venular RBC velocity, which was 0.84 ± 0.35 (MPA) and 1.02 ± 0.38 (MP4) of baseline.

The arteriolar and venular blood flows after hemodilution are presented in Fig. 3. The results are given as means ± SE to show the trend of this parameter calculated from vessel diameter and RBC velocity. Both arteriolar and venular blood flows were statistically increased from baseline after level 1 and level 2 exchange. Upon further hemodilution with Dx 70, HSA and MP4 arteriolar blood flow could not be sustained, whereas MPA maintained venular blood flow. Venular flow decreased for Dx 70 and HSA, whereas MPA and MP4 maintained venular flow. However, venular flow after level 3 exchange with MPA and MP4 was statistically significantly different form venular flows after level 3 exchange with Dx 70 and HSA.



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  		Fig. 3. Flow (nl/s, mean ± SD) in each animal group was as follows: baseline (A: 15.9 ± 4.1, n = 74, V: 6.0 ± 2.0, n = 78); level 1 with Dx 70 (A: 21.0 ± 5.7; V: 5.6 ± 2.4); level 2 with Dx 70 (A: 28.9 ± 7.6; V: 8.9 ± 4.2); level 3 with Dx 70 (A: 12.5 ± 5.3, n = 18, V: 2.7 ± 2.0, n = 16); level 3 with HSA (A: 10.9 ± 4.8, n = 16, V: 3.0 ± 2.3, n = 18); level 3 with MPA (A: 17.2 ± 6.6, n = 20, V: 5.8 ± 3.7, n = 20); level 3 with MP4 (A: 13.6 ± 6.0, n = 22, V: 7.2 ± 3.4, n = 22). P < 0.05 relative to baseline ({dagger}), compared with level 3 Dx 70 (§), and compared with level 3 HSA (¶).

 
Wall shear rate. The calculated wall shear rate in arterioles and venules is shown in Table 3. Tabulated results from the present study and previous reports show what appears to be a threshold of wall shear rate and correspondingly wall shear stress that is required for FCD to exceed 50% of baseline (Fig. 4).


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  		Table 3. Wall shear rate in arterioles and venules

 


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  		Fig. 4. Changes in arteriolar and venular vessel wall shear rate in arterioles and venules. Broken line is baseline. §P < 0.05 compared with level 3 Dx 70. Wall shear stress (dynes/cm2, mean ± SE) in each animal group was as follows: level 3 with Dx 70 (A: 5.6 ± 1.1, n = 18; V: 1.8 ± 0.8, n = 16); level 3 with HSA (A: 5.3 ± 1.3, n = 16, V: 1.5 ± 0.9, n = 18); level 3 with MPA (A: 6.2 ± 1.0, n = 20, V: 2.2 ± 1.1, n = 22); level 3 with MP4 (A: 6.6 ± 1.0, n = 20, V: 2.4 ± 1.2, n = 22).

 
FCD. FCD (Fig. 5) was reduced after level 1 exchange in all groups 91 ± 4% (P < 0.05 vs. baseline). Level 2 exchange reduced FCD to 83 ± 8% (P < 0.05 vs. baseline). FCD was further reduced after level 3 for all test solutions (Dx 70, 38 ± 16%, P < 0.05; HSA, 49 ± 14%, P < 0.05 vs. baseline; MPA, 67 ± 12%, P < 0.05 vs. baseline; MP4, 71 ± 9%, P < 0.05 vs. baseline).



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  		Fig. 5. Functional capillary density after the level 3 hemodilution for the different test fluids. All values are relative to baseline levels. P < 0.05 relative to baseline ({dagger}) and compared with level 3 Dx 70 (§).

 
Microvascular oxygen distribution. Oxygen tension measured using PQM after the exchange-transfusion with Dx 70, HSA, MPA, and MP4 showed that materials produced similar distributions of microvascular PO2 (arterioles averaged 38 mmHg and venules 3 mmHg; Fig. 6). Exchange with Dx 70 yielded an arteriolar PO2 of 32 mmHg, statistically significantly differences from the exchanges with MPA and MP4, whereas the venules had a PO2 similar for all the materials.



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  		Fig. 6. Intravascular partial oxygen pressure after level 3 exchange hemodilution with test materials. PO2 (mmHg, mean ± SD) in each animal group was as follows: level 3 with Dx 70 [A: 32.2 ± 3.8, n = 18; V: 3.3 ± 0.8, n = 16; tissue (T): 1.3 ± 1.2, n = 20]; level 3 with HSA (A: 34.7 ± 7.2, n = 16; V: 2.8 ± 0.8, n = 18; T: 1.7 ± 1.2, n = 20); level 3 with MPA (A: 42.4 ± 3.6, n = 20; V: 3.3 ± 0.9, n = 20; T: 1.9 ± 1.3, n = 20); level 3 with MP4 (A: 39.3 ± 4.6, n = 22; V: 2.7 ± 0.6, n = 22; T: 1.9 ± 1.2, n = 20); n, no. of vessels or locations studied. §P < 0.05 compared with level 3 Dx 70.

 
Tissue PO2 values in the level 3 exchange with Dx 70, HSA, MPA, and MP4 were 1.3 ± 1.2, 1.7 ± 1.2, 1.9 ± 1.3, and 1.9 ± 1.2 mmHg (not respectively statistical differences). According to previous studies in this species, normal tissue PO2 is 21.7 ± 3.5 mmHg (22; Fig. 6).

Oxygen delivery and extraction. Figure 7 shows the result of the analysis of oxygen delivery and release in the microcirculation. It is evident that exchanging RBCs for MP4 oxygen delivery and extraction to the tissue increased. The contribution of the remaining RBCs is the same for MP4 and MPA. However, MP4 apparently delivers all of its oxygen in the microcirculation, with the net result that oxygen release for MP4 is the same as for whole blood. Conversely, hemodilution with Dx 70 and HSA significantly reduces oxygen delivery by approximately one-fourth and extraction by the tissue by approximately one-third of baseline, respectively.



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  		Fig. 7. Arterial oxygen delivery (top) and extraction (bottom) before and after level 3 hemodilution. P < 0.05 relative to baseline ({dagger}), compared with level 3 Dx 70 (§), and compared with level 3 HSA (¶). Calculations of global oxygen transport are not directly measurable in our model. However, the changes relative to baseline can be calculated using the measured parameters. Briefly, a single animal oxygen delivery was calculated by averaging the product of oxygen content and relative flow of all arterioles. Similar calculation was performed using the information of the venules. The extraction was calculated as the difference between averaged arterioles and venules for each animal. Values of oxygen delivery and extraction include all animals within a group, reducing any bias. The difference in oxygen delivery and extraction between MP4 and MPA is statistically significant, P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
The principal finding of this study is that, in extreme hemodilution, PEG-conjugated albumin and Hb lead to physiological microvascular and systemic conditions that are closer to baseline than using non-PEG materials such as albumin and Dx 70. The principal systemic difference was that PEG materials caused CI to be significantly above baseline, and microvascular responses were equally more favorable, since arteriolar and venular blood flows were significantly increased relative to dextran and HSA (excepting arteriolar blood flow; Fig. 3). These effects were paralleled by changes in FCD where the use of PEG compounds caused this parameter to attain values significantly greater than those obtained using the conventional plasma expanders (Fig. 5).

The results obtained show that in terms of BE at Hct of 11% both PEG materials show no differences from the systemic conditions found at baseline, whereas the use of conventional plasma expanders shows a negative BE that is significantly different from baseline. COP attained at the end of the experimental period was significantly higher for the PEG materials (Table 4) than conventional plasma expanders, suggesting a lesser tendency to extravasation and more prolonged maintenance of blood volume. A difference between PEG compounds is apparent in Fig. 7, which shows that the additional oxygen-carrying capacity due to the presence of Hb of MP4 causes oxygen delivery and extraction in the microcirculation to be the same as in control conditions, and significantly higher than for MPA. The equivalence of MP4 and MPA at the level of hemodilution achieved in this study is in agreement with the results obtained in a study of 100% volume exchange using the same PEG materials in a rat exchange model (37) that showed that the difference in the ability to sustain the organism in terms of BE between compounds in rats only becomes apparent when Hct is reduced to 7.4%, which is a significantly lower threshold than that attained in our extreme hemodilution protocol, namely 11% (37).


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  		Table 4. Rheological properties and colloid osmotic pressure

 
The present study and the work of Winslow et al. (37) show that oxygen-carrying capacity is not the only prerequisite for maintaining the organism in extreme hemodilution. A similar study carried out using a polymerized bovine Hb (Oxyglobin; Biopure, Boston, MA) showed a significantly higher Hb plasma concentration than in the present study but did not result in a physiologically adequate FCD when used in the same extreme hemodilution protocol. This result was found to be the consequence of the vasoactivity of this material, which leads to lowering of capillary pressure and FCD (10, 11, 27).

Studies of extreme hemodilution with plasma expanders of different viscosities show that, at Hct 11%, Dx 70 and HSA that achieves plasma viscosities of 1.4 and 1.2 cP, respectively, cause FCD to fall below 50% of normal, and HES 200 (6%, hydroxyethyl starch 200 kDa in 0.9% sodium chloride) causes plasma viscosity to be 1.3 cP and FCD to 66% of baseline (8). By comparison, experiments with 500 kDa dextran and alginates caused plasma viscosity to be 2.2 and 2.7 cP, where FCD was 71 and 76%, respectively (9). In contrast, in the present experiments, plasma viscosity was 1.3 and 1.4 cP for MPA and MP4 and FCD was 62 and 65%, respectively. Plasma viscosity and vessel wall shear rate are determinants of vessel wall shear stress. Table 4 shows plasma and blood viscosities were not significantly different between the groups tested in this study and that the differences in shear stress appear to be primarily related to the differences in shear rate and therefore CI. Table 3 summarizes available data on blood and plasma viscosities, wall shear rate, and wall shear stress after extreme hemodilution. The relationship between plasma viscosity and FCD divides the compounds into the following groups: 1) low viscosity and low FCD (6% Dx 70 and HSA); 2) low viscosity and high FCD (all PEG compounds); and 3) high viscosity and high FCD (6% dextran 500 kDa, 10% HES 200 kDa, 0.7% alginate; see Refs. 8, 9, and 29).

Assuming none of the molecular species used as plasma expanders have any biological or chemical interaction with the microcirculation homeostasis (with the exception of MP4, which likely scavenges NO), the remaining possibility for interaction can be mechanic in terms of shear stress. The question arises whether these materials present the endothelium with a different level of shear stress, presumably leading to a difference in mechanotransduction on the endothelium, and, therefore, mediating a difference in production of the vasodilator by the endothelium. With the use of the data on shear rate given in Fig. 4, shear stress in dynes/cm2 x 102, (mean ± SE, n = no. of vessels studied in each animal group) at level 3 was as follows: Dx 70 [arterioles (A): 5.6 ± 1.1, n = 18; venules (V): 1.8 ± 0.8, n = 16]; HSA (A: 5.3 ± 1.3, n = 16, V: 1.5 ± 0.9, n = 18); MPA (A: 6.2 ± 1.0, n = 20, V: 2.2 ± 1.1, n = 22); and MP4 (A: 6.6 ± 1.0, n = 20, V: 2.4 ± 1.2, n = 22). Thus both MPA and MP4 produce somewhat higher levels of vessel wall shear stress than the conventional materials, although the difference is not significant.

The results on vessel wall shear stress, although approximate because of the uncertainty of the relevant viscosity used in the calculations, are related to the effect that shear stress has on perivascular NO concentration in hemodilution reported in the study by Tsai et al. (28). In the study of Tsai et al., with the use of conventional low-viscosity plasma expanders, microvascular flow was reduced, which correlated with decreased shear stress and lowered perivascular NO levels. The same effect on shear stress was found in the present study with conventional plasma expanders, as expected. PEG compounds did not significantly increase shear stress, suggesting that their action is not related to shear stress-mediated effects, such as NO production. Notably, MP4 is a scavenger of NO, having the same binding constant for NO as free Hb (23). However, MP4 effect on microvascular shear stress was identical to that of MPA, suggesting that the effects of MP4 are not related to NO management in the microcirculation.

As in previous studies with this protocol (12, 27, 32), in extreme hemodilution, tissue PO2 was very low, although in these studies in the case of MP4 the rate of oxygen release in the tissue was the same as baseline. This finding suggests that tissue PO2 is not a precise index of tissue metabolism. The significantly increased release of oxygen by MP4 relative to MPA shows that the high oxygen affinity of MP4 Hb does not impede the release of oxygen to the tissue. A previous study comparing oxygen release of MP4 vs. that of Oxyglobin (Biopure), a low-affinity material (P50 = 48 mmHg), showed that Oxyglobin used in the same protocol as the present study released oxygen prevalently in the premicrovascular circulation. In the present experiments, the release of oxygen by MP4 is estimated by determining the change of oxygen saturation between arterioles and venules for this compound, assuming MP4 PO2 values are the same as surrounding blood. This calculation shows that MP4 is 94% saturated in the arterioles and ~14% in the venules. Consequently, MP4 releases ~80% of its oxygen in the microcirculation. Because arterioles and venular PO2 are approximately the same for MPA and MP4, RBCs deliver the same amount of oxygen to both the pre- and microcirculatory compartments. Conversely, Dx 70 and HSA show significantly reduced oxygen delivery to the microcirculation, since arteriolar PO2 values are significantly lower relative to the PEG compounds.

Data on CO and MAP allow us to determine peripheral vascular resistance (PVR). This calculation shows that PVR in arbitrary units is 75 for dextran and 81 for HSA and drops to 54 for MPA and 58 for MP4. Thus the minimum decrease in PVR is 58/75 or ~23% (comparing MP4 and dextran) and a maximum of 34% comparing MPA and HSA.

Our results show that PEG-conjugated proteins have plasma expansion properties. During extreme hemodilution, PEG compounds produce an increase in CO, a condition difficult to achieve using a conventional colloid-based plasma expander. The elevated CO is accompanied by no statistically significant (P ~ 0.08) increase in HR, suggesting a somewhat increased stroke volume. The partially increased stroke volume leads to increased CO, which is facilitated by the decrease of PVR caused by PEG compounds. All of these findings suggest that the effect of PEG compounds compared with conventional plasma expanders is to increase both MAP and CO while lowering PVR. These effects are mirrored in the microcirculation where diameters and flows are generally greater for the PEG compounds than conventional plasma expanders.

In conclusion, our findings show that MPA and MP4 cause significantly increased CO in extreme hemodilution compared with the same level of hemodilution attained with conventional plasma expanders. Results may be in part due to the normalization of oxygen delivery to the heart, assuming that the microcirculation of the heart tissue responds to hemodilution in the same manor as the tissue in the chamber window model. The central effect of PEG compounds possibly would be an addition of greater vascular filling and decreased PVR, supported by the results where dextran and HSA were unsuccessful during extreme hemodilution. An important difference between PEG-based plasma expanders and conventional fluid is a significantly greater peripheral capillary perfusion (FCD). FCD coupled with high oxygen affinity should be the principal reason that allows PEG-Hb to carry oxygen more efficiently to the tissues, increasing oxygen extraction even though its plasma concentration is only 1% by weight.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
DISCLOSURES
 REFERENCES
 
This work was supported by the National Heart, Lung, and Blood Institute Bioengineering Partnership Grant HL-R24- 64395 and Grants HL-R01-40696, HL-R01-62354, and HL-R01-62318.


   DISCLOSURES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
REFERENCES
 
R. W. Winslow is president, chief executive officer, and chairman of the board of Sangart, Inc., San Diego, CA.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Cabrales, La Jolla Bioengineering Institute, 505 Coast Blvd. S., 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
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 

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