Am J Physiol Heart Circ Physiol
279: H908–H915, 2000.
Molecular dimensions of Hb-based O2 carriers determine
constriction of resistance arteries and hypertension
HIROMI SAKAI,1 HIROYUKI HARA,1 MINAKO YUASA,1 AMY G. TSAI,2 SHINJI TAKEOKA,1
EISHUN TSUCHIDA,1 AND MARCOS INTAGLIETTA2
1
Department of Polymer Chemistry, Advanced Research Institute for Science and Engineering,
Waseda University, Tokyo 169-8555, Japan; and 2Department of Bioengineering, University
of California, San Diego, La Jolla, California 92093-0412
Received 7 June 1999; accepted in final form 16 February 2000
(Hb) solutions, presently in advanced stages of clinical trials, may soon be
accepted as substitutes for blood transfusions in surgical procedures and treatment of trauma patients (49,
54). However, as clinical trials are extended to include
larger numbers of individuals, it becomes apparent
that the principal side effect consistently reported in
the administration of acellular Hb solutions is hypertension presumably because of vasoconstriction.
Hypertension, a well-defined reaction of the acellular
intramolecularly cross-linked Hb (XLHb), was proposed to be beneficial in the treatment of hypotension
concomitant to hemorrhagic shock (33). However, vasoconstriction reduces blood flow, lowering functional
capillary density, and therefore affecting tissue perfusion and oxygenation (10, 48). Furthermore, maintenance of functional capillary density per se, independently of tissue oxygenation, has been shown to be
critical to tissue survival in hemorrhagic shock (19).
Nitric oxide (NO) scavenging by Hb due to intrinsic
high affinity of NO to Hb is the mechanism presumed
to cause vasoconstriction and hypertension (7, 26, 45,
50). This theory was validated indirectly using exteriorized rabbit aortic rings in organ baths, where constriction was observed following the addition of acellular Hb solutions as well as an NO synthase inhibitor
(10, 34, 36). Different modifications of the Hb molecule
cause hypertension that is qualitatively and quantitatively different, and red blood cells (RBCs) and cellular
liposome-encapsulated Hb (Hb vesicles) do not cause
either vasoconstriction or hypertension (6, 36, 42).
Analysis of the reported data on different commercially developed Hb modifications suggests an inverse
relationship between Hb molecular size and the extent
of the pressor response, an effect that could be related
to the dynamics of NO-to-Hb interactions. To systematically explore this relationship, we produced O2-carrying Hbs of which their principal difference was their
molecular sizes. Because molecular size is a determinant of viscosity, we infused the solutions as a bolus of
10% of blood volume, thus not significantly changing
blood viscosity and shear stress.
Most evidence for the pressor response is obtained
from measurements of systemic pressure, and direct
evidence about the mechanism involved is scarce. In
previous studies in conscious hamsters fitted with a
dorsal skinfold, we found that small arteries of 130–
160 ␮m diameter, termed resistance vessels, exhibit
the greatest reactivity in hemorrhagic shock (39), playing a significant role in the regulation of blood flow.
Constriction of these resistance vessels in our model
was also directly correlated to the pressure response
following administration of NO synthase inhibitor (38).
Address for reprint requests and other correspondence: E.
Tsuchida, Dept. of Polymer Chemistry, Advanced Research Institute
for Science and Engineering, Waseda Univ., Tokyo 169-8555, Japan
(E-mail: [email protected]).
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.
blood substitutes; resistance vessels; microcirculation; autoregulation; nitric oxide; hemoglobin
ACELLULAR MODIFIED HEMOGLOBIN
H908
0363-6135/00 $5.00 Copyright © 2000 the American Physiological Society
http://www.ajpheart.org
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Sakai, Hiromi, Hiroyuki Hara, Minako Yuasa, Amy
G. Tsai, Shinji Takeoka, Eishun Tsuchida, and Marcos
Intaglietta. Molecular dimensions of Hb-based O2 carriers
determine constriction of resistance arteries and hypertension. Am J Physiol Heart Circ Physiol 279: H908–H915,
2000.—The effect of molecular dimension of hemoglobin
(Hb)-based O2 carriers on the diameter of resistance arteries
(A0, 158 ⫾ 21 ␮m) and arterial blood pressure were studied in
the conscious hamster dorsal skinfold model. Cross-linked
Hb (XLHb), polyethylene glycol (PEG)-conjugated Hb, hydroxyethylstarch-conjugated XLHb, polymerized XLHb, and
PEG-modified Hb vesicles (PEG-HbV) were synthesized.
Their molecular diameters were 7, 22, 47, 68, and 224 nm,
respectively. The bolus infusion of 7 ml/kg of XLHb (5 g/dl)
caused an immediate hypertension (⫹34 ⫾ 13 mmHg at 3 h)
with a simultaneous decrease in A0 diameter (79 ⫾ 8% of
basal value) and a blood flow decrease throughout the microvascular network. The diameter of smaller arterioles did not
change significantly. Infusion of larger O2 carriers resulted
in lesser vasoconstriction and hypertension, with PEG-HbV
showing the smallest changes. Constriction of resistance
arteries was found to be correlated with the level of hypertension, and the responses were proportional to the molecular dimensions of the O2 carriers. The underlying mechanism
is not evident from these experiments; however, it is likely
that the effects are related to the diffusion properties of the
different Hb molecules.
DIMENSIONS OF O2 CARRIERS AND VASOCONSTRICTION
In this study we aim to determine whether resistance vessel constriction is related to hypertension
after administration of acellular Hb and the extent to
which the effect is dependent on the size of acellular
Hb molecules modified by polymerization, polymer conjugation, and cellular liposome encapsulation.
MATERIALS AND METHODS
Polymerization of XLHb was carried out by step-wise addition of glutaraldehyde in an anaerobic condition for 8 h at
4°C. The unreacted glutaraldehyde was inactivated by excess
lysine. After carbonylation to stabilize Hb, unreacted Hb was
removed twice by salting out using ammonium sulfate (ionic
strength: 6 M). The obtained poly-XLHbCO was converted to
an oxy form and dialyzed against phosphate-buffered saline.
Cellular PEG-modified Hb vesicles (PEG-HbV) were prepared as previously reported (40). The vesicles contained Hb
(38 g/dl) and 6 mM of PLP as an allosteric effector. The
surface of HbV was modified with PEG (5,000 mol wt) by
mixing HbV suspension with a saline suspension of 1,2distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[polyethylene glycol]. The resulting PEG-HbV was ultracentrifugated to remove excess PEG-lipid and redispersed in a
sterilized phosphate-buffered saline. The suspension was
then filtered through sterilized filters (pore size: 0.45 ␮m).
Physicochemical characterization of Hb products. The Hb
preparations were characterized in terms of O2-binding properties, viscosity, diameter, molecular weight, oncotic pressure, contents of metHb, and monomeric Hb. Oxygen affinities (P50) were calculated from the O2 equilibrium curve
obtained with a Hemox Analyzer (TCS Medical Products).
Particle sizes were measured by light scattering (model
N4SD, Coulter particle analyzer). Oncotic pressure was measured with a model 4420, Wescor osmometer. Viscosity was
measured with a capillary viscometer (Oscillatory Capillary
Rheometer and Density Meter, OCR-D, Anton Paar, Austria)
at 37°C. Number average molecular weights (Mn) were calculated from oncotic pressure measurements (52). Weight
average molecular weights (Mw) were measured with a multiangle light scattering (miniDAWN DSP, Wyatt Technology)
equipped with a size exclusion column (Shodex Protein KW804). The presence of remaining monomeric XLHb in HESXLHb and poly-XLHb, and PLP-Hb in PEG-PLP-Hb was
detected by size exclusion chromatography (TOSO TSKgel
G3000SWXL), with an eluent of phosphate-buffered saline
(pH 7.4), a flow rate of 1 ml/min, and a detection wavelength
at 419 nm.
Animal model and preparation. Experiments were carried
out in 36 male Syrian golden hamsters weighing 66⫾ 8 g
(Simonsen, Gilroy, CA). All animals were housed in cages
and provided with food and water ad libitum in a temperature-controlled room on a 12:12 h light-dark cycle. After the
animals were anesthetized with intraperitoneally administered pentobarbital sodium (ca. 100 mg/kg body wt, Abbott,
North Chicago, IL), the dorsal skinfold consisting of two
layers of skin and muscle was fitted with two titanium
frames with a 15-mm circular opening and surgically installed. A location that included a paired small artery and
vein was selected. The resistance artery can be readily identified because a Y-shaped pair of artery and vein can be seen
visually when the hamster dorsal skin is extended after the
hair has been removed (39). Layers of skin muscle were
separated from the subcutaneous tissue and removed until a
thin monolayer of muscle, including the small artery and
vein, and one layer of intact skin remained. A cover glass
(diameter 12 mm) held by one frame covered the exposed
tissue allowing intravital observation of the small artery (A0,
diameter 158 ⫾ 21 ␮m), which is the main feeding vessel in
this tissue, large feeding arterioles (A1, 63 ⫾ 12 ␮m), and
small veins (V0, 364 ⫾ 73 ␮m).
Polyethylene tubes (PE-10, ca. 1 cm, Becton-Dickinson,
Parsippany, NJ) were connected to PE-50 (ca. 25 cm) via
silicone elastomer medical tubes (ca. 4 cm, Technical Products) and were implanted in the jugular vein and the carotid
artery. They were passed from the ventral to the dorsal side
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Preparation of chemically modified acellular Hbs. Hb was
purified from outdated donated blood obtained from the Hokkaido Red Cross Blood Center in Japan (40). The purification
procedure included carbonylation, heat treatment at 60°C,
ultracentrifugation (50,000 g, 40 min), ultrafiltration using
100-kDa membranes, and dialysis with 10-kDa membranes.
Carbonylhemoglobin is stable at 60°C so that the heat treatment enables denaturation of other concomitant proteins and
virus inactivation. Even though heating for 1 h is enough to
remove other proteins (41), the current heating period is 10 h
to ensure virus inactivation. This is the same condition for
the clinically used human serum albumin. The purified Hb
solutions were checked for the presence of residual phospholipids by extracting lipids with modified Bligh and Dyer’s
method and HPLC (41), where the removal efficiency was
⬎99.4%, the remaining phospholipid being ⬍4 ␮g/ml in the
purified 5% Hb solution. Endotoxin content was measured by
the conventional Limulus amoebocyte lysate test and was
found to be ⬍0.1 EU/ml in the Hb solution.
All Hb modifications were prepared with purified Hb obtained by the same procedure under sterile conditions (43), in
a class 10,000 clean room with clean benches equipped with
high-efficiency particulate air (HEPA) filters in which the
dust number is essentially zero. The room temperature was
regulated at 10–15°C. All instruments were autoclaved, and
the distilled water and saline used were of human injection
grade.
Intramolecularly XLHb was prepared according to Chatterjee et al. (4). DeoxyHb (1 mM, 6.45 g/dl) in the presence of
inositol hexaphosphate (5 mM, Sigma) was reacted with
(2,3-dibromosalicyl)fumarate (1.5 mM, Aldrich) and then
heated to 75°C to remove unreacted Hb and concomitant
proteins, which were then removed by ultracentrifugation
(19,000 g, 30 min). Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis confirmed the presence of ␣␣-dimers
(32,000 mol wt) and ␤-subunits (16,000 mol wt). Purified
XLHb was obtained after dialysis against phosphate-buffered saline (pH 7.4) and filtration through a sterilized filter
(pore size: 0.45 ␮m).
Polyethylene glycol (PEG)-conjugated pyridoxylated (pyridoxal 5⬘-phosphate, PLP) Hb (PEG-PLP-Hb) was prepared by
reacting pyridoxylated Hb with PEG (5,000 mol wt)-cyanuric
chloride (Sigma), which binds an amino group of Hb (16). The
unreacted materials were removed by ultra filtration using a
200-kDa membrane. This solution was then dialyzed and
suspended in a phosphate-buffered saline.
HES-XLHb was prepared using hydroxyethylstarch (HES,
70,000 mol wt, Kyorin, Tokyo, Japan) according to Tam et al.
(47) with some modification. One-third of the hydroxyl
groups were activated with cyanogen bromide (CNBr) and
then mixed anaerobically with deoxy-XLHb at 37°C. Amino
groups on the Hb molecule react with the activated HES to
form amide bonds. The unreacted sites were inactivated with
excess glycine. The crude Hb product was carbonylated and
purified twice by salting out using ammonium sulfate (ionic
strength: 6 M) to remove unreacted Hb and HES (11). The
obtained HES-XLHbCO was converted to an oxy form and
dialyzed against phosphate-buffered saline.
H909
H910
DIMENSIONS OF O2 CARRIERS AND VASOCONSTRICTION
on-line in arterioles and venules (15). Diameter was measured with an image-shearing system (Digital Video Image
Shearing Monitor 908, IPM, San Diego, CA), whereas RBC
velocity was analyzed by photodiode and the cross-correlation technique (Velocity Tracker Mod-102 B, IPM). Blood flow
rates (Q̇) were calculated by means of Eq. 1
Q̇ ⫽ ␲(RBC velocity/R v)(diameter/2) 2
(1)
where Rv is the ratio of velocity in the middle of vessels to
whole blood velocity. We selected Rv ⫽ 1.6 according to
Lipowsky and Zweifach (22), who specified this value for
vessels from 13 to 90 ␮m diameter, but not for larger vessels.
Because all changes are normalized relative to the basal
values, the results are not affected by the value of Rv selected.
Data analysis. Differences between treatment groups were
analyzed using a one-way ANOVA followed by Fisher’s protected least significant difference test. A paired t-test was
used to compare the time-dependent changes within each
group. The changes were considered statistically significant
if P ⬍ 0.05. Changes in MAP and HR are expressed as
differences from the basal value. Changes in microvascular
diameter and blood flow rates were expressed as the percentage of basal values.
RESULTS
Physicochemical characteristics of Hb-based O2 carriers. The physicochemical characteristics of the Hb
molecules in solution used in these experiments are
presented in Table 1. Their molecular diameters were
7 ⫾ 2 for XLHb, 22 ⫾ 2 for PEG-PLP-Hb, 47 ⫾ 17 for
poly-XLHb, 68 ⫾ 24 for HES-XLHb, and 224 ⫾ 76 nm
for PEG-HbV. XLHb (5 g/dl) had an oncotic pressure of
15.8 mmHg. Polymerization of XLHb reduced this to
2.5 mmHg at the same concentration. PEG-PLP-Hb
had the largest oncotic pressure (70.2 mmHg) and the
highest viscosity (6.1 cP at 332 s⫺1) due to the highly
hydrated PEG chains (52), whereas that of PEG-HbV
was close to zero because the number of particles in
suspension is significantly reduced. The larger particle
size of HES-XLHb (68 ⫾ 24 nm) and higher oncotic
pressure than poly-XLHb are due to the additional
highly hydrated HES chains and more expanded struc-
Table 1. Properties of Hb-based oxygen carriers at [Hb] ⫽ 5 g/dl
Parameters
Diameter by Coulter analyzer, nm
Mn, kDa
Mw, kDa
[Hb], g/dl
[HES, PEG, or lipid], g/dl*
P50, mmHg
Hill number
[metHb], %
Monomeric Hb, %
Oncotic pressure, mmHg
Viscosity at 332 s⫺1, cP
Half-life in Wistar rats, h
XLHb
7⫾2
72
66
5
0
32
2.0
2.2
15.8
1.0
1.6 (4)†
PEG-PLP-Hb
Poly-XLHb
HES-XLHb
PEG-HbV
22 ⫾ 2
186 (123)*
189
5
4.5
14
1.3
4.5
4.4
70.2
6.1
47 ⫾ 17
510
2,154
5
0
20
2.2
8.0
2.7
2.5
1.5
3
68 ⫾ 24
431
2,782
5
3.4
22
2.2
4.5
3.3
9.5
2.2
224 ⫾ 76
5
2.9
18
2.1
2.5
0
2.6
4 (30)†
Hb, hemoglobin; XLHb, cross-linked Hb; PEG-PLP-Hb, poly(ethylene glycol)-modified Hb containing pyridoxal 5⬘-phosphate; HES,
hydroxyethyl starch; HbV, Hb vesicles; Mn, number average molecular weight; Mw, weight average molecular weight. Mn was calculated with
colloid osmotic pressure. Mw was calculated with light-scattering analysis. * The weights of HES and PEG were obtained by gravinometry
of dried samples. Half-life in Wistar rats after infusion in Wistar rats (10% blood volume overdose). † 20% blood volume overdose of samples
with 10 g/dl Hb concentration.
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of the neck and exteriorized through the skin at the base of
the chamber. The patency of the catheters was ensured by
filling with heparinized saline (40 IU/ml).
Microvascular observations of the awake and unanesthetized hamsters were performed at least 5 days after chamber
implantation to mitigate postsurgical trauma. During the
measurements the animals were placed in a perforated plastic tube, from which the window chamber protrudes, to minimize animal movement without impeding respiration. A
preparation was considered suitable for experimentation if
microscopic examination of the window chamber met the
criteria of no sign of bleeding and/or edema and the diameter
of the resistance vessel was larger than 130 ␮m. After surgery the A0 arteries were constricted to ⬍130 ␮m; however,
after 4 or 5 days they recovered and were about 150 ␮m in
average diameter. Hamsters with A0 arteries below 130 ␮m
were excluded from the study because they might not have
completely recovered from the surgical intervention.
All animal studies were approved by the Animal Subject
Committee of University of California, San Diego, and performed according to National Institutes of Health Guidelines
For The Care And Use Of Laboratory Animals (NIH publication 85-23, Revision 1985).
Infusion of Hb samples. The infusion volume was 7 ml/kg,
which is 10% of the total blood volume of a hamster (70 ml
blood/kg). Therefore, the amount of Hb infused was 350
mg/kg because all Hb solutions had [Hb] ⫽ 5 g/dl ([heme] ⫽
3.1 mM). Six experiments were made with each material.
Human serum albumin solution (HSA, 5 g/dl, Bayer) was
used as a control. Sample solutions of ⬃0.4 ml were infused
through the jugular vein at a rate of 0.4 ml/min. The catheter
was flushed with a small amount of saline.
Characterization of systemic conditions. Mean arterial
pressure (MAP) and heart rate (HR) were recorded in analog
format (Beckman R611, Beckman Instruments, Schiller
Park, IL) from a transducer connected via an arterial catheter.
Microhemodynamic analysis. The microvasculature was
observed with an inverted microscope (IMT-2, Olympus, Tokyo, Japan) using a ⫻10 objective (Olympus) and a ⫻40
water immersion objective (Olympus, Wplan) and transillumination. Microscopic images were recorded by video (Cohu
4815–2000, San Diego, CA) and transferred to a TV-VCR
(Sony Trinitron PVM-1271Q monitor, Tokyo, Japan) and a
Panasonic AG-7355 video recorder (Tokyo, Japan). Microvascular diameter and center-line RBC velocity were analyzed
DIMENSIONS OF O2 CARRIERS AND VASOCONSTRICTION
H911
Fig. 2. Changes in diameter (A) and blood flow rates (B) in the
resistance artery (A0) after infusion of different Hb products. The
time point 0 h indicates ⬍30 s after completion of infusion. Values
are means ⫾ SE. *Significantly different from baseline. #Significantly different from the PEG-HbV group. The baselines of diameter and
blood flow rate were 158 ⫾ 21 ␮m and 316 ⫾ 250 nl/s, respectively.
Fig. 1. Changes in mean arterial pressure (MAP, A) and heart rate
(HR, B) after infusion of different hemoglobin (Hb) products (HSA,
human serum albumin; HES, hydroxymethyl starch; XLHb, crosslinked Hb; PEG, polyethylene glycol; HbV, Hb vesicles, PLP, pyridoxal 5⬘-phosphate). The time point 0 h indicates ⬍30 s after completion of infusion. Values are means ⫾ SE. *Significantly different
from baseline. #Significantly different from the PEG-HbV group. The
baselines of MAP and HR were 106 ⫾ 8 mmHg and 414 ⫾47
beats/min, respectively.
tended to decline after infusion; the heart rate of the
XLHb group decreased by 84 ⫾ 44 beats/min 2 h after
infusion. The PEG-HbV group exhibited the same level
of bardycardia as the HSA group.
Microvascular responses. Significant constriction
was observed in A0 of resistance arteries (Fig. 2) but
not in smaller arterioles (A1) nor capacitance venules
(V0). For example, the diameters (% of basal value) of
A1 and V0 in each of the groups after 2 h were as
follows: XLHb (97⫾5 and 99 ⫾4), PEG-PLP-Hb (101
⫾4 and 102 ⫾1), poly-XLHb (106 ⫾10 and 100 ⫾3),
HES-XLHb (101 ⫾2 and 104 ⫾2), and PEG-HbV (101
⫾4 and 103 ⫾4). The changes in diameter of the A0
vessels and MAP were plotted against the molecular
sizes of Hb samples (Fig. 3). Vasoconstriction, which
was most significant in the XLHb group, decreased
with increasing molecular size; the HES-XLHb group
showed results similar to those of the PEG-HbV group.
Blood flow rates of the XLHb group declined significantly in A0 vessels (36 ⫾17% of basal value at 3 h) and
tended to return to control values after 24 h. The
PEG-HbV and HSA groups showed small flow and A0
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ture. The Mw and Mn of poly-XLHb and HES-XLHb are
significantly different due to the wide molecular weight
distribution.
XLHb had the lowest O2 affinity (highest P50, 32
mmHg). Polymer conjugation and polymerization of
XLHb increases O2 affinity (decreases P50) of Hb. The
P50 of PEG-HbV (18 mmHg) was regulated by coencapsulating PLP. The content of metHb in all samples was
less than 8% of the total Hb. Trace amount of monomeric Hb was present in the polymer-conjugated and
polymerized Hbs. The remaining monomeric Hb content was 4.4% for PEG-PLP-Hb, 2.7% for poly-XLHb,
and 3.3% for HES-XLHb.
Systemic responses. Control MAP for all groups was
106 ⫾ 8 mmHg. The XLHb group increased blood
pressure immediately after infusion (⫹12 ⫾ 8 mmHg
relative to baseline), which was sustained for 3 h
(⫹34 ⫾ 13 mmHg) (Fig. 1). This effect subsided after
24 h. The HSA and PEG-HbV group had no significant
changes. The PEG-PLP-Hb and poly-XLHb groups had
intermediate responses. Heart rates of all the groups
H912
DIMENSIONS OF O2 CARRIERS AND VASOCONSTRICTION
diameter changes. The remaining Hb groups showed
intermediate changes depending on their sizes. The A0
vessels of the poly-XLHb group constricted reaching
87 ⫾ 7% of the basal value at 3 h, and their flow
reduced to 54 ⫾16% at 2 h. The HES-XLHb group
showed the smallest changes of all modified Hbs, and
effects were similar to those of the PEG-HbV group.
DISCUSSION
Our principal finding is that the hypertensive effect
following the administration of acellular Hb solutions
is directly correlated to vasoconstriction of the resistance arteries (A0 vessels) and that the magnitude of
the effect is an inverse function of the molecular size of
the material. Vasoconstriction in our model was circumscribed to the A0 vessels and did not extend to the
remainder of the microvasculature.
Our findings agree with the concept that the resistance arteries are as important as the arteriolar network in regulating peripheral blood flow (27). This is
supported by studies in hypertensive rats where large
arterioles and small arteries, and not small arterioles,
are responsible for the increase in total organ vascular
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Fig. 3. Influence of particle diamters of Hb products on MAP (A) and
A0 diameter (B). The time point 0 h indicates ⬍30 s after completion
of infusion. The data are converted from Figs. 2 and 3.
resistance (2, 25, 31). Similarly, in previous studies in
our model of conscious hamsters we found that A0
resistance arteries are the most reactive in hypotensive shock (39). In a recent study, we infused 10 mg/kg
of the NO synthase inhibitor N␻-nitro-L-arginine
methyl ester (L-NAME) and found the immediate onset
of hypertension (⫹22 mmHg) and simultaneous constriction of A0 vessels (⫺30%), whereas the downstream arterioles A1 –A3 did not constrict (38). Thus we
can presume that the resistance arteries are the vessels most sensitive to NO and crucial in determining
blood pressure and flow when NO production is inhibited. Vasoconstriction induced by the NO synthase
inhibitor NG-monomethyl-L-arginine was reported for
dog coronary artery, human brachial artery, and hamster cheek pouch arterioles (26), all of which are resistance vessels. Stewart et al. (46) reported the constriction of coronary resistance vessels in the isolated
rabbit heart by native Hb. Griffith et al. (12) showed
that rabbit ear small arteries (diameter range 150–700
␮m), where resistance and shear stress are maximal,
have the highest sensitivity to endothelium-derived
relaxation factor.
Although studies using aortic rings demonstrated
vasoactivity of Hb and the NO synthase inhibitor (29,
36), it was recently reported (3) that the aorta in
anesthetized rabbits did not constrict after the infusion
of the NO synthase inhibitor L-NAME or dextran-conjugated Hb. Thus the peripheral arteries, and not the
aorta, constrict to induce hypertension. Nolte et al. (32)
reported that arterioles smaller than 60 ␮m in diameter did not remain constricted even though hypertension was long lasting after infusion of XLHb. These
results indirectly support the concept that resistance
arteries between the aorta and arterioles are crucial in
Hb-induced vasoconstriction and resulting hypertension.
The vasoconstrictive responses in our study may be
in part explained in terms of NO scavenging by Hb (13)
and the relative capacity of the different molecules to
extravasate. Nakai et al. (29) has shown that the molecular size of Hb products is a factor in determining
their passage across the endothelial cell layer. In this
context, the smallest sized XLHb would be the most
permeable and would show a higher level of vasoconstriction and hypertension than PEG-Hb and liposomeencapsulated Hb. Smaller Hb molecules may extravasate in greater numbers and thus bind a greater
amount of NO, whereas the larger HbV particles with
diameters of about 250 nm would extravasate in significantly fewer numbers. Poly-XLHb and HES-XLHb,
which are larger than XLHb, caused less vasoconstriction and correspondingly an intermediate level of hypertension, as shown in Fig. 3.
A large pressor effect was recently reported for 50%
exchange transfusions of Dex-BTC-Hb, a combination
of dextran and Hb with a 300-kDa mean molecular
mass (8). This effect was found to correlate with the
rapid penetration of Hb in the endothelium, although it
was not determined whether Hb or Dex-BTC-Hb had
penetrated these cells. It should be noted that the
DIMENSIONS OF O2 CARRIERS AND VASOCONSTRICTION
increase the rate of NO binding, lowering the availability of NO to the smooth muscle. Verification of this
mechanism requires measurement of the NO reaction
rate with the different Hbs used.
Measurements by the flash photolysis method show
that modified Hb molecules with molecular dimensions
ranging from normal Hb to that of PEG-conjugated Hb
have similar Hb-NO binding rates (about 3.0⫻107
M⫺1s⫺1 in Ref. 35); however, the binding rate of larger
size molecules was not determined. The characteristic
of PEG-conjugated Hb is that the PEG chains are
highly hydrated, resulting in a large excluded volume
and a significantly larger molecular diameter than
XLHb. However, the NO-binding rate measurement
indicated that NO molecules can diffuse in the PEGwater layer to bind Hb without any hindrance. In our
study we compared the binding rate of unmodified Hb
and Hb vesicles by the stopped flow method and we
found the binding rate constants to be 3⫻107 M⫺1s⫺1
and 4.8⫻106 M⫺1s⫺1, respectively (37), indicating that
the surface-to-volume ratio of the particles plays a role
in determining the NO-trapping rate; thus Hb encapsulation may significantly contribute to retarding NO
binding by the same mechanism that retards O2 binding (5). The O2 binding rate is related to particle size
(53), and that of HbV is one order of magnitude slower
than acellular Hbs, although faster than RBCs. Thus
the vasoconstrictive effect does not appear to be explained solely on the basis of NO-binding rates, because the rates of HbV and RBC are different. However, neither of them causes vasoconstriction.
Therefore, the relationship between the molecular dimension and permeability across the endothelial cell
layer should be considered in parallel.
In our experiment the amount of the infused Hb was
one-thirtieth of blood Hb, and the solution volume was
10% of blood volume to minimize the effects due to
changes in viscosity, oncotic pressure, and O2 affinity.
Viscosity is governed by the RBC concentration, and
this small dilution did not affect blood viscosity and
therefore shear stress on the vascular wall. PEGPLP-Hb has an oncotic pressure of 70.2 mmHg, which
may have increased blood oncotic pressure by about 7
mmHg, possibly causing a small increase in vascular
volume through autotransfusion.
Remaining phospholipids and endotoxin contamination during Hb purification may cause hemodynamic
effects; however, endotoxin contamination reduces
blood pressure (20). Macdonald et al. (23) reported
unpurified stroma-free Hb induced more significant
coronary vasoconstriction than purified Hb. We determined the phospholipid and endotoxin concentrations
in the purified Hb solution but not in the final Hb
solutions. However, all the materials were prepared
from the same purified Hb; thus the response due to
XLHb infusion is unlikely to be due to a different level
of contamination. Gulati et al. (14) have shown that
stroma free Hb with phospholipid contamination of
more than 50 ␮g/ml induced renal failure, although
hypertension was reduced; however, our samples
should be significantly below this value.
Downloaded from http://ajpheart.physiology.org/ by 10.220.32.247 on June 15, 2017
molecular weight of Dex-BTC-Hb is much smaller than
that of HES-XLHb and poly-XLHb in our study. PEG
conjugation of XLHb resulted in reduced hypertensive
responses in rats according to Abassi et al. (1), although a similar product has been proposed for the
treatment of septic shock to increase blood pressure
(9). Glutaraldehyde-polymerized bovine Hb and o-raffinose-polymerized human Hb, which are currently under clinical trials, are also reported to induce hypertension (17, 35). The molecular dimensions of these
products are much smaller than those of HES-XLHb
and PEG-HbV in our study.
We measured the plasma retention half-life of XLHb,
poly-XLHb, and PEG-HbV at the same dosages in
Wistar rats (Table 1). XLHb had a half-life of 1.6 h,
whereas poly-XLHb and PEG-HbV had half-lives of 3
and 4 h, respectively. The shorter half-life of XLHb
suggests a greater tendency for extravasation and
therefore an increased potential for inducing vasoconstriction. About 20% of XLHb remained in the circulation after 3 h and was not detectable after 8 h, which
was consistent with the observation that hypertension
lasted for 3 h and was not present after 24 h.
Duration and the level of vasoconstriction and hypertension may be partly related to the concentration
of Hb in plasma (24). In some reports an immediate
response after the infusion of Hb was observed, although the maximal effect occurred 0.5 or 1.0 h after
infusion, even though the XLHb concentration was the
highest just after the infusion (24, 45). In our experiments peak hypertension was seen at 3 h and peak
vasoconstriction was at 2 h after infusion; however,
there was no significant difference among 1, 2, and 3 h.
Schultz et al. (45) have shown that significant hypertension was maintained over 1.5 h at 280 mg/kg infusion of XLHb in rats, which correlates with our findings
in which 350 mg/kg of XLHb showed hypertension for
3 h. Our results agrees with those of Malcolm et al.
(24), who indicated that the bolus infusion in 10 s
induced a longer and higher level of hypertension than
a slower 4-min infusion. We infused XLHb in 1 min,
which may explain the duration of hypertension. Isolated femoral arterial strip experiments show that a
Hb concentration of 1 ␮M is enough to induce vasoconstriction (18). In our experiment about 20% of infused
XLHb remains in the blood after 3 h (⬃16 ␮M); therefore, the sustained hypertension and vasoconstriction
may be related to the presence of Hb.
An alternative explanation is that the scavenging
effect may be operational directly on the blood side of
the endothelium, where the presence of molecular Hb
in the RBC-free plasma layer could significantly distort
the diffusion field from the endothelial cell, diverting
NO from smooth muscle into blood according to
Vaughn et al. (51). In normal blood there is an RBCfree layer plasma region next to the endothelium, and
in this region NO is not consumed. For the vessels in
our study, this region is ⬃2.5-␮m thick (44), and the
presence of this layer introduces a resistance to the
diffusion of NO to the site of scavenging the RBCs. The
presence of Hb in this plasma layer should significantly
H913
H914
DIMENSIONS OF O2 CARRIERS AND VASOCONSTRICTION
The authors acknowledge to Dr. P. C. Johnson (Dept. Bioengineering, Univ. of California, San Diego) and Dr. D. Erni (Inselspital
University Hospital, Bern) for discussion on resistance artery and Y.
Mano, M. Hamasaki, H. Onuma, and K. Tomiyama (Waseda Univ.)
for the Hb samples preparation. H. Sakai was a research fellow of the
Japan Health Sciences Foundarion. E. Tsuchida is a Core Research
for Evolutional Science and Technology investigator, JSTC.
This work has been supported in part by USPHS/NHLBI Program
Project Grant HL-48018; Grants in Aid for Scientific Research from
the Ministry of Education, Science, Sports, and Culture, Japan
(12480268); and the Health Science Research Grants (Research on
Advanced Medical Technology, Artificial Blood Project, H10-Blood007), the Ministry of Health and Welfare, Japan.
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