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Am J Physiol Heart Circ Physiol 285: H2543-H2551, 2003. First published July 24, 2003; doi:10.1152/ajpheart.00537.2003
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O2 release from Hb vesicles evaluated using an artificial, narrow O2-permeable tube: comparison with RBCs and acellular Hbs

Hiromi Sakai,1 Yoji Suzuki,2 Megumi Kinoshita,2 Shinji Takeoka,1 Nobuji Maeda,2 and Eishun Tsuchida1

1Advanced Research Institute for Science and Engineering, Waseda University, Tokyo 169-8555; and 2Department of Physiology, School of Medicine, Ehime University, Shigenobu, Ehime 791-0295, Japan

Submitted 11 June 2003 ; accepted in final form 21 July 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURE
 REFERENCES
 
A phospholipid vesicle that encapsulates a concentrated hemoglobin (Hb) solution and pyridoxal 5'-phosphate as an allosteric effector [Hb vesicle (HbV) diameter, 250 nm] has been developed to provide an O2 carrying ability to plasma expanders. The O2 release from flowing HbVs was examined using an O2-permeable, fluorinated ethylenepropylene copolymer tube (inner diameter, 28 µm) exposed to a deoxygenated environment. Measurement of O2 release was performed using an apparatus that consisted of an inverted microscope and a scanning-grating spectrophotometer with a photon-count detector, and the rate of O2 release was determined based on the visible absorption spectrum in the Q band of Hb. HbVs and fresh human red blood cells (RBCs) were mixed in various volume ratios at a Hb concentration of 10 g/dl in isotonic saline that contained 5 g/dl albumin, and the suspension was perfused at the centerline flow velocity of 1 mm/s through the narrow tube. The mixtures of acellular Hb solution and RBCs were also tested. Because HbVs were homogeneously dispersed in the albumin solution, increasing the volume of the HbV suspension resulted in a thicker marginal RBC-free layer. Irrespective of the mixing ratio, the rate of O2 release from the HbV/RBC mixtures was similar to that of RBCs alone. On the other hand, the addition of 50 vol% of acellular Hb solution to RBCs significantly enhanced the rate of deoxygenation. This outstanding difference in the rate of O2 release between the HbV suspension and the acellular Hb solution should mainly be due to the difference in the particle size (250 vs. 7 nm) that affects their diffusion for the facilitated O2 transport.

blood substitutes; red blood cells; hemoglobin; microcirculation; oxygenation; liposome

VARIOUS KINDS OF HEMOGLOBIN (Hb)-based O2 carriers (HBOCs) have been developed for the substitution of the function of red blood cells (RBCs) including intramolecularly cross-linked, polymerized, polymer-conjugated, and liposome-encapsulated Hbs (LEHs; Refs. 6, 42). Historically, it has been regarded that the O2 affinity (expressed as P50; the O2 tension at which Hb is half-saturated with O2) should be regulated similar to that of RBCs, namely, ~25–30 Torr, using an allosteric effector or by a direct chemical modification of the Hb molecules. Theoretically, this allows sufficient O2 unloading during blood microcirculation as can be evaluated by the arteriovenous difference in O2 saturation (SO2) in accordance with an O2 equilibrium curve. It has been expected that decreasing O2 affinity (increasing P50) results in an increase in O2 unloading. This is supported by the result in a mouse model (31) that RBCs with a high P50 value show enhanced O2 release for improved exercise capacity.

The size of HBOCs is much smaller than that of RBCs. Thus even if the O2 affinity of both O2 carriers is similar in the equilibrated condition, the O2 releasing and binding rates should be different depending on the flow conditions of the carriers and the diffusion of O2. In a stopped-flow analyses, significantly faster O2 release rates for an unmodified Hb solution or LEHs rather than RBCs were confirmed (e.g., O2 dissociation rate constant, 84 s–1 for unmodified Hb solution vs. 4.4 s–1 for RBCs; Refs. 8, 30, 45). However, these observations were performed under homogeneous conditions and at dilute (<10 µM) Hb concentrations ([Hb]), and it was not clear whether this significant difference would actually be observed in the in vivo condition and how the microcirculation would respond to the difference.

O2 transfer from blood to tissues in the microcirculatory network is the result of a complex process whereby a substantial fraction of O2 is exchanged in the arterioles and venules (10, 11, 14) where RBCs are not homogeneously distributed. RBCs tend to move to the centerline in laminar flow, and there is a plasma layer in the marginal zone as is clearly demonstrated in microvessels (17). For this reason, studies of O2 release in microvessels as well as in capillaries are physiologically important (14, 34, 38, 39, 43). Therefore, the O2 release rates under more physiological conditions, at a higher [Hb], and in flow conditions in microvessels have been required for discussion of the dynamics of the O2 releasing that couples with tissue oxygenation. The measurement can be performed using an artificial, narrow, O2-permeable tube (35). In the case of blood, a cell-free plasma layer between a RBC-flow column and a vessel wall and a highly viscous Hb solution inside RBCs could be barriers to the O2 diffusion. On the other hand, HBOCs are so small that they are homogeneously dispersed in the plasma phase. Page et al. (20) demonstrated using an artificial flow channel excavated in an O2-permeable silicone rubber film that a polymerized bovine Hb showed facilitated O2 release when the Hb was mixed with RBCs.

Phospholipid vesicles that encapsulate concentrated Hb have been developed [Hb vesicles (HbVs) or LEHs; diameter, 250 nm] as another type of HBOC that possesses a cell structure similar to RBCs (21, 24–30, 42). Both RBCs and HbVs have a lipid-bilayer membrane that prevents direct contact of Hb with blood components and endothelial lining. Furthermore, Hb encapsulation in the vesicle suppresses hypertension induced by vasoconstriction; this mechanism is presumably due to the effects of free Hb, which scavenges the endogenous vasorelaxation factors, nitric oxide, and carbon monoxide (13, 24) due to their high affinity with Hb. In this report, we evaluate for the fist time (based on microscopic observations) the O2 unloading profile of our HbVs compared with RBCs and an acellular unmodified Hb solution using an artificial, narrow, O2-permeable tube with a 28-µm inner diameter. This methodology has been well established by Tateishi et al. (35, 36).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURE
 REFERENCES
 
Preparation of HbVs, Hb solutions, and an RBC suspension. The HbVs were prepared under sterile conditions as previously reported (24, 27, 32, 33). Hb was purified from outdated donated blood obtained from the Hokkaido Red Cross Blood Center (Sapporo, Japan) and the Japanese Red Cross Society (Tokyo). The encapsulated purified Hb (38 g/dl) contained 17.6 mM of pyridoxal 5'-phosphate (PLP; Sigma; St. Louis, MO) as an allosteric effector at a molar PLP/Hb ratio of 2.5. The lipid bilayer was composed of a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, cholesterol, and 1,5-O-dihexadecyl-N-succinyl-L-glutamate in a 5:5:1 molar ratio (Nippon Fine Chemical; Osaka, Japan). The surface of the HbVs was modified with polyethylene glycol (PEG; 5,000 mol wt) using 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-polyethylene glycol (NOF; Tokyo; 0.3 mol% of total lipids). HbV diameter was 252 ± 53 nm (determined by the light-scattering method). The P50 value was 28 Torr at 37°C, which was measured with a Hemox analyzer (TCS Medical Products; Huntingdon Valley, PA). HbVs were resuspended in either 5 g/dl recombinant human serum albumin (HSA; 69,000 mol wt; Nipro; Osaka; HbVHSA) or in 6 g/dl hydroxylethyl starch (HES; 70,000 mol wt; Kyorin Chemistry Lab; Tokyo; HbVHES), and the final [Hb] was adjusted to 10 g/dl.

The purified Hb solution (38 g/dl) was diluted with a phosphate-buffered saline to 10 g/dl, and PLP was added at a molar Hb/PLP ratio of 1:3 or 1:1. Under these conditions, the P50 values were 29 and 15 Torr, respectively, and these Hb solutions were termed Hb29 and Hb15, respectively. Fresh human RBCs were obtained from a healthy donor (Y. Suzuki). RBCs were washed twice with isotonic saline and suspended in the saline that contained 5 g/dl HSA, and the [Hb] was adjusted to 10 g/dl. The RBC suspension was used within a day of the blood collection. The physicochemical properties of all O2 carriers are summarized in Table 1. The viscosities of the suspensions were measured with either a capillary rheometer (oscillatory capillary rheometer OCR-D; Anton Paar; Graz, Austria) or a cone-plate viscometer (model E; Tokyo Keiki; Tokyo) at 37°C and at the shear rates of 300 and 71 s–1. The latter shear rate is approximately identical to the wall shear rate of the narrow tube (inner diameter, 28 µm) as performed in the present study when the centerline velocity is 1 mm/s. The HbV or Hb solutions were mixed with the RBC suspension at volume ratios of 0:100, 10:90, 50:50, 90:10, and 100:0. For example, the mixture of 10 vol% HbVHSAs and 90 vol% RBCs is abbreviated as 10HbVHSA/90RBC for convenience.


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  		Table 1. Physicochemical properties of Hb solutions and HbV and RBC suspensions

 

Perfusion of HbV/RBC or Hb/RBC mixtures through narrow tubes. Narrow, O2-permeable tubes (inner diameter, 28.1 or 28.6 µm; wall thickness, 37.5 µm; length, 150 mm) were produced from a fluorinated ethylenepropylene copolymer at Hirakawa Hewtech (Ibaraki, Japan) as described by Kubota et al. (16). One end of the narrow tube was connected to a reservoir of the HbV/RBC or Hb/RBC suspension. The narrow tube was placed horizontally between two wide acrylic plates with a short gap (~2 mm) on the stage of an inverted microscope (IMT-2; Olympus Optics; Tokyo). The suspension in the reservoir was gently and continuously mixed with a magnetic stirrer. The gap between the two acrylic plates was filled with nitrogen-bubbled saline that contained 10 mM sodium hydrosulfite (Na2S2O4; Wako Pure and Fine Chemical; Tokyo). The narrow tube was not permeable to sodium hydrosulfite. The HbVs distributed homogeneously in the narrow tube so that it was difficult to determine the marginal HbV-free layer. Therefore, the thickness of the marginal RBC-free layer, the distance between the tube inner wall and a nearest RBC, was measured at 50 points using an image-processing technique (35), and the values were averaged. The perfusion pressure was monitored with a sensor (P-231D; Nihon Kohden; Tokyo) equipped with an amplifier (AP-601G; Nihon Kohden). The entire perfusion experiment was performed at 25°C.

Measurement of O2 in narrow tubes. The apparatus (35) consisted of an inverted microscope with an objective lens of x40 magnification (ULWD CDPlan 40PL; Olympus Optics), a scanning-grating spectrophotometer with a sensitive photon-counting detector that counted 1 x 103 to 3 x 106 photons/s (USP-410; Unisoku; Osaka), and a computer (PC-386V; Epson; Tokyo). The light intensity of a halogen lamp in the microscope was controlled by a current stabilizer (NLO 18-10; Takasago; Tokyo). The spectrophotometer was connected to the microscope eyepiece through a thin light guide (diameter, 0.4 mm) made of quartz and was operated by the computer. The grating was scanned in 508 steps over the wavelength range of 499.2–600.8 nm with a gate time of 20 ms/step for photon counting; data were obtained every 0.2 nm. One visible spectrum from a 5-µm-diameter spot over the centerline of the narrow tube was recorded within 20 s. A measuring spot on the narrow tube within the visual field of the microscope was fixed on a monitor (PVM-1371; Sony; Tokyo) through a video camera attached to the vertical eyepiece (DXC-930; Sony) by sliding the stage of the microscope and/or by rotating the cylindrical mirror. The centerline flow velocity was determined using the cross-correlation technique (2, 15), and the velocity was adjusted to 1.0 mm/s by changing the pressure applied to the reservoir of HbV/RBC or Hb/RBC mixtures. Under these conditions, the Reynolds number was 0.025 and the laminar flow was theoretically established.

The spectroscopic measurements were performed at traveling distances of 15, 30, 50, 80, and 100 mm. After a steady flow of RBCs and a steady oxygenation state of the flowing RBCs and HbVs (or Hbs) were attained a few minutes later, 10 scans were accumulated. Numerical filtering was then applied with a moving average of 5 successive values (e.g., absorbances at 499.6, 499.8, 500.0, 500.2, and 500.4 nm) in the scanning step to obtain a smoothed absorption spectrum (used to improve the signal-to-noise ratio). One scan of RBCs flowing in the narrow tube showed a remarkable deviation in absorbance values (Fig. 1A). On the other hand, HbVs showed less deviation due to the homogeneous dispersion of the vesicles. The accumulation of 10 scans followed by the moving averaging of 5 successive values in the scanning step of every 0.2 nm provided a smooth spectrum even for the RBC suspension (Fig. 1B). However, owing to the light scatter by fine particles, the absorbance of the HbVs at a shorter wavelength was slightly higher than that at a longer wavelength (29).



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  		Fig. 1. A: one scan of the red blood cell (RBC) suspension showed a significantly scattered spectrum, whereas hemoglobin (Hb) vesicles (HbVs) showed a smooth spectrum. B: accumulation of 10 scans followed by the moving average of 5 successive values (using a 0.2-nm scanning step) provided enough resolution to calculate the O2 saturation (SO2). C: for SO2 determination, two isobestic points of the spectra of deoxygenated (deoxy-) and oxygenated (oxy)HbVs (at 522 and 586 nm) were connected by a straight line (baseline). On the basis of the absorbances at 555 ({Delta}A555, {lambda}max of deoxyHb) and 576 ({Delta}A576, {lambda}max of oxyHb) nm from the baseline, a relationship between SO2 and the ratio of the two absorbances (R = {Delta}A555/{Delta}A576) was expressed as SO2 (in %) = (35 – 15R)/(0.25R + 0.21).

 

In the spectra of the 100%-deoxygenated and 100%-oxygenated HbVs, two isosbestic points (522 and 586 nm; Ref. 44) in the Q band of Hb were connected by a straight line as the baseline (Fig. 1C). The absorbances at 555 nm ({Delta}A555, {lambda}max of deoxyHb) and 576 nm ({Delta}A576, {lambda}max of oxyHb) from the baseline were obtained to make a calibration line that shows the relationship between the O2 (in %) and the ratio of the two absorbances (R = {Delta}A555/{Delta}A576) as O2 = (35 – 15R)/(0.25R + 0.21). The O2 values of each sample were plotted versus the traveling distance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURE
 REFERENCES
 
Flow patterns of HbV/RBC mixtures in narrow tube. Figure 2 shows a microscopic view of the mixtures of RBCs and HbVHSAs flowing in the narrow tubes. The thickness of the RBC-free marginal layer increased with increasing mixing ratio of HbVHSA, and the layer seemed to be slightly turbid and dark colored due to the presence of HbV particles with a 250-nm diameter (29). The thickness of the RBC-free layer was 2.7 ± 1.7 µm for RBCs alone (100RBC), 3.5 ± 1.8 µm for 10HbVHSA/90RBC, 4.8 ± 2.2 µm for 50HbVHSA/50RBC, and 7.0 ± 1.6 µm for 90HbVHSA/10RBC. On the other hand, the mixture of RBCs and the Hb solution produced a transparent layer, but the distribution of RBCs was not changed significantly compared with the HbV/RBC mixtures (data not shown).



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  		Fig. 2. Flow patterns of RBCs mixed with HbVs suspended in recombinant human serum albumin (HbVHSA/RBC) in a narrow tube. HbV particles were homogeneously dispersed in a suspension medium. They tended to distribute in the marginal zone of the flow. Thickness of the RBC-free layer increased with increasing amount of HbVHSA. RBC-free phase became darker and more semitransparent, which indicates the presence of HbVs. Tube diameter, 28 µm; Hb concentration ([Hb]), 10 g/dl; centerline flow velocity, 1 mm/s.

 

Perfusion pressure of narrow tube. The perfusion pressure of the RBC suspension mixed with HbVHSA, HbVHES, or Hb29 in various ratios was examined at the constant centerline flow velocity of 1 mm/s (Fig. 3). The addition of the Hb29 solution to the RBC suspension in HSA (3.9 cP; Table 1) decreased the perfusion pressure from 10 kPa (100RBC) to 6 kPa (90Hb29/10RBC) due to the lower viscosity of the Hb solution (1.3 cP at 71 s–1). On the other hand, the addition of HbVs increased the perfusion pressure in proportion to the HbV/RBC mixing ratio. Especially, the perfusion pressure (41 kPa) of the mixture of 90% HbVHESs (5.7 cP; Table 1) and 10% RBC (3.9 cP) was more than four times higher than that of RBCs alone (10 kPa).



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  		Fig. 3. Perfusion pressure measurements of RBCs mixed with Hb-VHSA (HbVHSA/RBC), hydroxylethyl starch (HbVHES/RBC), or purified Hb and added pyridoxal 5'-phosphate (PLP), which has a P50 value of 29 Torr (Hb29/RBC). [Hb] flowing in the narrow tube, 10 g/dl; centerline flow velocity, 1 mm/s.

 

Spectroscopic changes of HbV/RBC and Hb/RBC mixtures. Figure 4 shows the representative spectroscopic changes of the mixtures of RBCs with HbVs or Hbs at various traveling distances in the narrow tubes. The most significant change was observed for the mixture of 90 vol% Hb29 solution and 10 vol% RBCs (90Hb29/10RBC). Two characteristic peaks of oxyhemoglobin in the Q band significantly decreased with traveling distance, and at 100 mm, the two peaks were flattened. On the other hand, the 90HbVHSA/10RBC mixture showed slight changes, but the changes were almost identical to those observed for the 100RBC suspension. The addition of 10 vol% Hb29s to 90 vol% RBCs (to make 10Hb29/90RBC) did not seem to significantly change the spectrum.



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  		Fig. 4. Representative spectroscopic changes in Q bands of 100% RBCs and various HbVHSA/RBC and Hb29/RBC mixtures compared with traveling distance in the narrow tube. Mixture of 90% Hb29 with 10% RBCs (90Hb29/10RBC) showed significant reduction in the two characteristic peaks attributed to oxyHb. Mixtures shown: 90HbVHSA/10RBC, 90% HbVHSA mixed with 10% RBCs; 10Hb29/90RBC, 10% Hb29 mixed with 90% RBCs.

 

O2 release from HbV/RBC mixtures. According to the calibration line for the SO2, the SO2 values (in %) were plotted vs. the traveling distances. Figure 5 summarizes the O2 release from the HbV/RBC mixtures in various mixing ratios. The rates of O2 release from all the mixtures were similar to that of 100RBC, and ~40% of the O2 was released at the traveling distance of 100 mm.



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  		Fig. 5. O2 release from the HbVHSA/RBC mixture for different mixing ratios determined at various traveling distances.

 

The influence of the suspending medium of HbV on O2 release was compared using HbVHSA and HbVHES in the mixture of 90 vol% HbVs and 10 vol% RBCs (Fig. 6). Irrespective of the suspending medium (HSA and HES), the rates of O2 release were almost identical for the two mixtures.



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  		Fig. 6. Effects of suspending media on O2 release from HbV/RBC mixture: comparison of 90HbVHSA/10RBC and 90% HbVHES with 10% RBCs (90HbVHES/10RBC) determined at various traveling distances.

 

O2 release from Hb/RBC mixtures. Contrary to the results with the HbV/RBC mixtures, the Hb/RBC mixtures showed faster O2 release rates than RBCs alone at the mixing ratio of 50 vol% for Hb29 and 90 vol% for both Hb29 and Hb15 (Fig. 7). The Hb29 (P50 = 29 Torr) possessed a similar P50 value as HbV (P50 = 30 Torr; Table 1). The 10Hb29/90RBC mixture did not show any apparent change in the O2 release compared with RBCs alone. However, the addition of 50Hb29/50RBC clearly showed a faster O2 release. The 90Hb29/10RBC mixture showed a much faster release rate, and 55% of the O2 was released at the 100-mm traveling distance. Addition of Hb15, which has a higher O2 affinity (P50 = 15 Torr), also enhanced the O2 release, but the enhancement was less than that of Hb29 as evaluated for the 50Hb15/50RBC mixture. However, at the mixing ratios of 10 and 90 vol%, there were no apparent differences in enhancement of O2 unloading between Hb15 and Hb29.



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  		Fig. 7. A: effects of O2 affinity on O2 release from Hb/RBC mixtures at various traveling distances: comparison of Hb29/RBC and Hb15/RBC mixtures of various ratios. B: effects of Hb/RBC or HbV/RBC mixing ratios on SO2: data from A for Hb/RBC and from Fig. 5 for HbV/RBC at the traveling distance of 100 mm. Hb15, purified Hb with pyridoxal 5'-phosphate (PLP) added, which has a P50 value of 15 Torr.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURE
 REFERENCES
 
The principal finding of this study using the narrow O2-permeable tube is that the rate of O2 release from HbVs is similar to that from RBCs at a constant [Hb] of 10 g/dl. On the other hand, acellular, unmodified Hb showed faster O2 release rates. This property of HbVs is outstanding compared with previous findings for O2 release rates measured by the stopped-flow method. The stopped-flow analysis confirmed that HbVs released O2 significantly faster than RBCs and slower than acellular, unmodified Hbs (30). Vandegriff and Olson (45) also examined the O2 release of RBCs of different sizes (diameter, 4.32–50 µm) from various species and of LEHs (diameter, 200 nm, which is similar in size to HbVs). They clarified that the O2 release rate depended primarily on the cell surface area-to-volume ratio and the intracellular [Hb] and that LEHs had the fastest release rate. These discrepancies of the results between the perfusion study using the O2-permeable tube and the stopped-flow methods should be mainly due to the differences in the experimental conditions. The stopped-flow studies were performed with homogeneous solutions at the low [Hb] of 10 µM (0.065 g/dl) in a mixing cuvette in which the dissociated O2 immediately diminished with sodium hydrosulfite and the O2 gradient between intra- and extracellular compartments was extremely high. In the present study using the O2-permeable tube, the [Hb] was as high as 1.6 mM (10 g/dl) with a high viscosity, and a certain kind of O2 gradient between the center of the tube and the tube wall would be formed. Therefore, the present experimental conditions were much closer to the in vivo physiological conditions, although the wall of the narrow tube was unavoidably thick.

We confirmed under our experimental conditions that acellular, unmodified Hb shows a much faster O2 unloading rate than RBCs as reported by other researchers (18, 20). The differences in the rates between the acellular Hb and HbVs could be explained by the theory of facilitated O2 transport by the diffusion of O2-bound Hb molecules (1, 5, 7). The total O2 flux is the sum of the fluxes of free O2 molecules and O2 bound as HbO2. The diffusion of HbO2 contributes to the facilitated O2 transport. According to the Stokes-Einstein equation, the diffusion constant (DHbO2) is defined as

(1)
where k is the Boltzman constant and T is absolute temperature. This equation demonstrates that the diffusion of a macromolecule, i.e., the contribution of facilitated O2 diffusion, is inversely proportional to the viscosity of the solution ({eta}) and the radius of the macromolecule (r); therefore, the contribution of facilitated O2 diffusion is inversely proportional to the size of the carriers and the viscosity (18).

Nishide et al. (19) studied O2 diffusion across a nonflowing solution membrane, and they found that the presence of Hb molecules in a solution (<30 g/dl) significantly facilitated O2 transport. When comparing HbVs, unmodified human Hb (HbAo), and polymerized Hbs, the "apparent" DHbO2 values are in the following order: HbAo (9 x 10–7 cm2/s) > polyHbs (2 x 10–7 cm2/s) > HbVs (3.5 x 10–8 cm2/s) at [Hb] = 10 g/dl, with solution viscosity values (1.0 < 1.8 < 3.2 cP, respectively) and molecular sizes (7 < ~15 < 200 nm, respectively) that are in inverse order in accordance with Eq. 1. As a result, facilitated O2 transport is significantly reduced for the suspension of HbVs.

According to the study of McCarthy et al. (18), a PEG-conjugated Hb (PEG-Hb) shows a similar O2 release rate with RBCs in an artificial microflow channel, whereas the acellular cross-linked Hbs and HbAo show faster rates. The PEG chains should not be a barrier layer to the O2 release. The main difference in the O2 release between PEG-Hb and other acellular Hbs should be due to the diffusion of Hb and the viscosity: PEG-Hb has a larger molecular size (28 vs. 7 nm) and a higher viscosity (3.2 vs. 0.9 cP) than the Hb molecule. These properties of PEG-Hb may reduce the O2 unloading, because the diffusion constant of PEG-Hb should be lower than that of an unmodified Hb. The higher O2 affinity of PEG/Hb (P50 = 10 Torr) should also contribute to the slower O2 unloading, because we confirmed that the Hb15/RBC mixture showed a slower O2 unloading than did the Hb29/RBC mixture at a 50:50 mixing ratio. Attention should be paid to the theory that facilitated O2 transport ([O2] facilitated) decreases with increasing P50 according to (1, 19)

(2)
where [Hb]0 is the total [Hb], PO2 is the O2 partial pressure, DHb is the diffusion coefficient of Hb, and K is the equilibrium constant of the O2 binding reaction (its reciprocal is proportional to the P50 value). Accordingly, the faster O2 release rate for Hb29 than for Hb15 is not due to the higher level of facilitated O2 transport but is simply a result of the lower O2 affinity.

When a large amount of HbV is exchange transfused, the addition of a plasma expander is required, because the oncotic pressure of the HbV suspension is close to zero (26). HbVHES shows a higher viscosity (5.7 cP at 71 s–1) compared with HbVHSA (3.9 cP), because the interaction of HbV particles with extended HES chains is possibly stronger than that with globular HSA molecules. Using the optical microscope, however, we could not observe the HbV aggregates in the suspension; this was possibly due to the low molecular weight of HES (70,000). The marginal RBC-free layer formed by axial accumulation of RBCs is important for the maintenance of blood flow as a lubricating layer (3). The addition of HbVHSA to RBCs increased the thickness of the RBC-free layer from 2.7 ± 1.7 (100RBC) to 7.0 ± 1.6 µm (90HbVHSA/10RBC). This increase was due to preferential flow of smaller particles, HbVs, near the peripheral region of the flow channel (4) and reduction of RBCs (i.e., reduction of hematocrit) (31). Furthermore, the perfusion pressure was increased at a constant flow rate, because the addition of the viscous HbVHES and HbVHSA suspensions increased the viscosity of the RBC-free lubricating layer. The increase in the perfusion pressure was more evident for the more viscous HbVHES than for HbVHSA possibly owing to the differences in the molecular conformations of HES and HSA; also, differences in their concentrations also partly contributed to the perfusion pressure values (37). Despite the viscosity difference between the two suspensions, O2 release rates from the RBC mixtures were not significantly altered in the present experimental conditions. It has been confirmed (35) that the diffusion constant of the O2 molecule is not significantly influenced by medium viscosity. The medium viscosity may influence the flow behavior of HbVs in a narrow tube. However, a homogeneous distribution of HbVHES and HbVHSA in the narrow tube was observed by cross-sectional scanning of the narrow tube during flow, and the vesicle-free layer was hardly observed in the marginal region. Therefore, homogeneous distribution of HbVs in the narrow tube and the identical O2 affinity of the vesicles provided identical O2 release rates from HbVHES and HbVHSA under a constant flow velocity. This phenomenon also supports the fact that the contribution of HbVs to facilitated O2 transport by diffusion of HbVs is significantly lower than acellular Hbs; thus viscosity does not influence O2 unloading. We conclude that under the present experimental conditions, differences in medium viscosity affect the perfusion pressure but not the O2 release from HbVs and/or RBCs.

The low contribution of HbVs to facilitated O2 transport could explain the similar O2 release rates for HbVs (250 nm) and RBCs (8 µm). Under conditions with little facilitated O2 transport for both HbV and RBC suspensions, only the O2 affinity (P50 value) would be the determining factor for the O2 release rate, because the P50 values for HbVs and RBCs were the same (28 Torr) in the present study. This speculation has to be confirmed in a future study of HbVs and RBCs with different P50 values.

Experiments of Page et al. (experiment A; Ref. 20) and ours (experiment B) showed the enhancement of O2 unloading by addition of acellular Hb solutions. However, there are some discrepancies between the two groups. Experiment A showed that the addition of only 10 vol% of Hb solution to the RBC suspension enhanced O2 release, whereas in experiment B, significant enhancement was observed with addition of 50 vol% of Hb solution, and enhancement was hardly observed for addition of 10 vol% of Hb solution. Furthermore, the reduction of SO2 in experiment A was much faster than in experiment B despite the identical Hb concentration (10 g/dl) and similar tube diameter (25 µm). For example, the 60% SO2 level of RBCs was attained after 100 s and 100 mm of traveling in experiment B, whereas the same SO2 level was attained after <0.2 s and only 4 mm of traveling distance in experiment A; the flow velocity was much faster in experiment A (~30 mm/s) than in experiment B (1.0 mm/s).

These differences between experiments A and B may be explained as follows. 1)O2 permeability of microflow channels: the silicone tube has a higher O2 permeability [~1,000–6,000 x cm3(STP) · cm–2 · cm–1 · s–1 · mmHg–1 x 1010] compared with the fluorinated ethylenepropylene copolymer tube [59 x cm3(STP) · cm–2 · cm–1 · s–1 · mmHg–1 x 1010] used in experiment B. Thus the silicone tube extracts O2 from the RBC suspension very rapidly by exposure to the deoxygenated environment. 2) Temperature: experiment A was conducted at 37°C, whereas experiment B was performed at 25°C. The higher temperature results not only in a higher P50 value (22) but also in more facilitation of the process of O2 unloading in experiment A. 3) Flow velocity: measurements in experiment B were carried out when the RBC flow and oxygenation attained steady state at a flow velocity of 1 mm/s. However, the measured SO2 of the flowing RBCs at an observation point showed the RBCs to be in the process of O2 unloading. In this condition, the measured SO2 would be higher than in the RBC-O2 equilibrium state. The O2 distribution in the tube has a parabolic shape; therefore, O2 is transferred from the centerline toward the wall of the tube. Such an O2 gradient in the tube may be more prominent at a higher flow velocity and with higher O2 permeability of the tube as in experiment A, as there would be less time for O2 unloading. Conversely, the thicker marginal RBC-free layer formed at the faster flow velocity may make the Hb-induced facilitation of O2 release more evident. 4) Measurement of SO2: in experiment B, SO2 was measured on a 5-µm-diameter spot over the centerline of the narrow tube in which O2 was retained for a longer traveling distance, whereas in experiment A, the SO2 was measured for the entire section of the flow channel. However, we confirmed that the SO2 at the center was not significantly different from that at a point a half-radius distance from the center after 100 mm of travel. In relation to the wavelength for the spectroscopy, the measurement in the Soret band (400–450 nm) in experiment A was remarkably influenced by the light scattering of the RBCs. On the other hand, the measurement in the Q band with a longer wavelength in experiment B was less influenced by the light scattering. These differences in spectroscopy do not seem to provide any quantitative differences in the O2 release rate. In conclusion, the quantitative difference between O2 release in experiments A and B is mainly due to the difference in the O2 permeability of the flow channels and partly due to the differences in temperature and flow velocity.

It has been suggested that faster O2 unloading from the HBOCs is advantageous for tissue oxygenation (20). However, this concept is controversial with regard to the recent findings, because an excess O2 supply would cause autoregulatory vasoconstriction and microcirculatory disorders (1, 23, 41). We confirmed that HbVs do not induce vasoconstriction and hypertension. This is not only owing to the reduced inactivation of nitric oxide as an endothelium-derived vasorelaxation factor (24) but also possibly due to the moderate O2 release rate that is similar to RBCs as confirmed in this study. Very recently, Erni et al. (9, 12) demonstrated that HbVs suspended in dextran effectively oxygenated ischemic collaterized tissue in skin flaps. This phenomenon should be explained by the functional characteristics of HbVs; namely, HbV suspension continues to retain O2 in upstream vessels and reaches the ischemic tissue to release the O2 (9, 12). In this regard, it is expected that HbVs with a lower P50 value would show much slower O2 unloading, which would be advantageous for ischemic tissue oxygenation. Moreover, the higher viscosity and the resulting higher perfusion pressure (as shown in Fig. 3) would be beneficial to increase the shear stress on the vascular wall for vasorelaxation and to homogeneously transmit the pressure to the microvascular network and thus supply blood to whole capillaries (39). Our experimental results contribute importantly to the design and optimization of O2 carriers and suggest the possible utilization of HbVs for new clinical indications other than blood substitution.


   DISCLOSURE
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURE
REFERENCES
 
This work was supported by Health Sciences Research Grants (Research on Pharmaceutical and Medical Safety, Artificial Blood Project); the Ministry of Health, Labor, and Welfare, Japan, and Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (B12480268); and 21 COE, Practical NanoChemistry, from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.


   ACKNOWLEDGMENTS
 
The authors are grateful to Dr. N. Tateishi (Ehime University) for setting up the microscope for the measurements and for the computer programming and to Dr. M. Intaglietta (University of California, San Diego) for promoting this study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Tsuchida, Advanced Research Institute for Science and Engineering, Waseda Univ., Tokyo 169-8555, Japan (E-mail: eishun{at}waseda.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURE
 REFERENCES
 

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