From the Department of Medicine, School of Medicine,
University of California, San Diego, Veterans Affairs Medical
Center, San Diego, California 92161 and ¶ Department of Chemistry
and Biochemistry, University of California, San Diego, La Jolla,
California 92093
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ABSTRACT |
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Changes in mean arterial pressure were monitored
in rats following 50% isovolemic exchange transfusion with solutions
of chemically modified hemoglobins. Blood pressure responses fall into
three categories: 1) an immediate and sustained increase, 2) an
immediate yet transient increase, or 3) no significant change either
during or subsequent to exchange transfusion. The reactivities of these hemoglobins with nitric monoxide (·NO) were measured to test the
hypothesis that different blood pressure responses to these solutions
result from differences in ·NO scavenging reactions. All
hemoglobins studied exhibited a value of 30 µM1 s
1 for both
·NO bimolecular association rate constants and the rate
constants for ·NO-induced oxidation in vitro. Only
the ·NO dissociation rate constants and, thus, the equilibrium
dissociation constants varied. Values of equilibrium dissociation
constants ranged from 2 to 14 pM and varied inversely with
vasopressor response. Hemoglobin solutions that exhibited either
transient or no significant increase in blood pressure showed tighter
·NO binding affinities than hemoglobin solutions that exhibited sustained increases. These results suggest that blood pressure increases observed upon exchange transfusion with cell-free hemoglobin solutions can not be the result of ·NO scavenging reactions at
the heme, but rather must be due to alternative physiologic
mechanisms.
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INTRODUCTION |
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Control of blood pressure and resistance to blood flow is achieved by a dynamic constriction and relaxation of smooth muscle tissue which surrounds all blood vessels except capillaries. Vascular smooth muscle tension is continually adjusted by a complex system that causes either vasoconstriction or vasodilation, depending on metabolic need (1). Research performed over the last decade has established that endothelium-derived nitric oxide (·NO)1 can cause vasodilation. ·NO is produced by endothelial cells that lie between the intravascular space and the surrounding smooth muscle. Among the findings was the demonstration that ·NO donors (e.g. nitroprusside, nitroglycerin) lead to vasorelaxation through activation of guanylate cyclase, whereas inhibitors of ·NO synthesis (e.g. NG-monomethyl-L-arginine) or scavengers (e.g. hemoglobin) cause vasoconstriction (for reviews, see Refs. 2 and 3).
Since cell-free hemoglobin is being developed as a red cell substitute (4), reactions between hemoglobin and ·NO are of potential importance in maintenance of microvascular blood flow and O2 delivery. Despite the wide variation that exists in the physical properties (O2 affinity, molecular mass, and solution properties) of different cell-free hemoglobins, it appears vasoconstriction is a feature common to many hemoglobin solutions (for reviews, see Refs. 2, 3, and 5). It is tempting to conclude that ·NO scavenging is the principal, if not sole mechanism for vasoconstriction associated with cell-free hemoglobin. However, it is well established that multiple factors contribute to the physiological control of vascular smooth muscle tone, and other mechanisms may operate as well (6).
Recently, cell-free hemoglobin solutions that differ in blood pressure
response were described by Migita et al. (7). These workers
compared bovine hemoglobin chemically modified by surface conjugation
to polyethylene glycol (PEG-Hb) with human hemoglobin cross-linked
between the lysine 99 residues of the subunits (
-Hb).
Isovolemic exchange transfusions in rats with a solution of
-Hb
resulted in a significant increase in MAP, whereas PEG-Hb solutions
caused no significant change in blood pressure. If increases in MAP are
due to ·NO scavenging reactions, then these two hemoglobins
would be expected to exhibit different reactivities with ·NO. We
have made a direct test of the hypothesis that the blood pressure
response to cell-free hemoglobin is related to the reactivity of
hemoglobin with ·NO by studying the vasopressor response to
hemoglobin solutions and the rate constants for reactions with
·NO. In addition to
-Hb and PEG-Hb, we have studied four
other chemically modified hemoglobins (see Table I). The list includes examples of the three principle types of hemoglobin modification under
development for use as a red cell substitute: intramolecular cross-linking, polymerization, and surface conjugation.
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EXPERIMENTAL PROCEDURES |
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Hemoglobin Solutions--
Chemically modified hemoglobin
preparations were dissolved in Ringer's lactate to make the solutions
used for both exchange transfusion experiments and in vitro
kinetic studies. Human hemoglobin cross-linked with
bis-(3,5-dibromosalicyl) fumarate between the chains at lysine 99 (
-Hb, 7.9 g/dl, 4.9 mM heme) was supplied as a gift
from the U.S. Army, Blood Research Detachment, Walter Reed Army
Institute for Research (8). Purified native human hemoglobin
(HbA0, 9.0 g/dl, 5.5 mM heme) was supplied as a
gift from Hemosol, Inc. Human hemoglobin cross-linked with trimesoyl tris (methyl phosphate) to make either a two-point intramolecular cross-link between the
chains at lysine 82 (
82-Hb, 6.8 g/dl, 4.3 mM heme) or a three-point cross-link between the
chains at lysine 82 and valine 1 of one
chain (Tm-Hb, 7.0 g/dl,
4.4 mM heme) were prepared as described by Kluger et
al. (9) and supplied as a gift from Hemosol, Inc. Human hemoglobin
polymerized with ring-opened raffinose (o-R-poly-Hb, 9.4 g/dl, 5.9 mM heme) was also supplied as a gift from
Hemosol, Inc. (10). Human hemoglobin modified by covalent attachment of
pyridoxal-5'-phosphate and surface conjugation to
-carboxymethyl,
-carboxymethoxypolyoxyethylene (PHP, 7.7 g/dl, 4.8 mM
heme) was supplied as a gift from Apex Biosciences, Inc. (11). Bovine
hemoglobin surface conjugated to methoxypolyoxyethylene glycol (PEG-Hb,
5.5 g/dl, 3.4 mM heme) was supplied as a gift from Enzon,
Inc. (12).
MAP Responses to 50% Isovolemic Exchange Transfusion-- Measurements of mean arterial pressure were conducted using groups of male Sprague-Dawley rats (250-350 g, Charles River Labs). All animal protocols were approved by the Animal Care Committee of the San Diego Veterans Affairs Medical Center. Animals were fed ad libitum prior to all experiments. Surgical preparation was performed 1 day prior to the exchange transfusion experiment. Rats were anesthetized with 250 µl of a mixture of ketamine (71 mg/ml; Aveco Co., Fort Dodge, IA), acepromazine (2.85 mg/ml; Fermenta, Kansas City, MO), and xylazine (2.85 mg/ml; Lloyd Laboratories, Shenandoah, IA). The areas of the femoral arteries and veins were exposed by blunt dissection. A specially constructed polyethylene catheter (PE-10 connected to PE-50) was placed into the abdominal aorta via one femoral artery to allow rapid withdrawal of arterial blood. An identical catheter was placed in the femoral artery of the opposite leg to monitor blood pressure. A PE-50 catheter was placed in the inferior vena cava via a femoral vein to allow infusion of test solutions. Catheters were tunneled subcutaneously, exteriorized through the tail, held in place by a plastic sheath assembly, and flushed with approximately 100 µL of normal saline. Animals were allowed to recover from the surgical procedure and remained in their cages for an additional 24 h before being used in experiments.
On the day of an exchange transfusion experiment, a conscious rat was placed in a plastic restrainer. The arterial and venous cannulae were flushed with 200 and 100 µl, respectively, of heparinized saline (100 units/ml). The venous catheter and the arterial catheter used for withdrawal were connected to a peristaltic pump (Labconco model 4262000, Kansas City, MO). The arterial catheter implanted to monitor blood pressure was connected to a pressure transducer (UFI model 1050, Morro, CA). Arterial pressure was sampled continuously at 100 Hz using a MP100WSW data collection system (BIOPAC Systems, Inc., Goleta, CA). The data were stored in digital form for subsequent off-line analysis. Animals were monitored for 30 min to acquire base-line data. An isovolemic exchange transfusion to 50% of total blood volume (calculated as 1/2 × 0.065 × g of body mass) was then performed with a hemoglobin test solution. The pump was operated so that blood was removed at exactly the same rate as test material was infused (0.5 ml/min). The duration of the exchange depended on the volume infused and lasted between 20 and 30 min. Test solutions were filtered through a 0.22-µm filter immediately prior to infusion, and the infusate tubing was passed through a 37 °C water bath. Systolic and diastolic pressures were the maximum and minimum pressures, respectively, and the mean arterial pressure (MAP) was calculated as diastolic + (systolic-diastolic)/3. Values of mean arterial pressure were averaged for each minute of data collected at 100 Hz. Statistical analyses were done using PROPHET software (BBN, Cambridge, MA). Errors were estimated as the standard error of the mean. Changes in MAP were considered significant based on the t test when p < 0.05.Measurement of Oxygen Equilibrium Curves--
Hemoglobin-oxygen
equilibrium curves were measured as described by Vandegriff et
al. (13). Hemoglobin solutions (0.1 M bis-Tris propane, 0.1 M Cl, pH 7.4) were rapidly
deoxygenated using the protocatechuic acid/protocatechuic acid
3,4-dioxygenase system (14). Both the enzyme and substrate were
obtained from Sigma. The deoxygenation reaction was followed by
simultaneous measurements of hemoglobin visible spectra and pO2 using a Milton Roy 3000 diode array
spectrophotometer (SLM Instruments, Inc., Urbana, IL) and a
micro-oxygen electrode (Microelectrodes, Inc., Londonderry, NH). Data
were analyzed as hemoglobin fractional saturation versus of
pO2 and fitted for Adair constants,
p50, and Hill's coefficient at 50% saturation
(n50).
Sample Preparation for in Vitro ·NO Reaction
Studies--
All gases were obtained from Matheson. Sodium
hydrosulfite (sodium dithionite) was provided as a gift from
Hoechst-Celanese. Sodium chloride, and bis-Tris propane were obtained
from Sigma. Anaerobic buffers were prepared by bubbling the solutions
with either nitrogen or argon. ·NO gas was scrubbed before use
by bubbling through anaerobic 2 M potassium hydroxide
solution to remove contaminating products of ·NO autoxidation.
The scrubbed ·NO gas was flushed through a glass tonometer
equipped with input and output stopcocks and a side port that was
sealed with a rubber septum. After flushing the tonometer with
·NO gas, anaerobic 0.1 M bis-Tris propane, 0.1 M Cl, pH 7.4, buffer was added to the
tonometer through the septum. The buffer was equilibrated under 1 atm
of ·NO gas to produce [·NO] = 1.97 mM stock
solutions. Solutions at lower ·NO concentrations were prepared
by dilution of a stock ·NO solution with the appropriate volume
of anaerobic buffer.
Kinetic Measurements-- Bimolecular association kinetics were measured by laser flash photolysis, a technique which takes advantage of the photosensitivity of the bond between ferrous heme protein and ligand (15). A Lumonics XeCl Excimer (308 nm) laser pumped a dye laser to produce a light pulse 4 ns in duration that averages 3 mJ per pulse at 540 nm. The observation light was provided by a tungsten-halogen lamp, and both the photolysis and observation beams impinged from counterpropagating directions onto a 2-mm path length cuvette containing the Hb-NO sample. ·NO recombination kinetic transients were monitored at 435 nm. Substantially less than 1% photolysis was produced by 100% laser light intensity due to the extremely low quantum yield for dissociation of Hb-NO complexes (15), and 1000 transients were averaged for each sample to improve the signal-to-noise ratio and produce usable ·NO recombination time courses. The time courses were fitted to a single exponential expression, taking into account the shape of the laser pulse and the response time of the instrument, using an iterative non-linear least squares algorithm to obtain the observed rate constants (kobs). Under these conditions, the reaction is pseudo first order and the bimolecular association rate constants were obtained by dividing the observed rate constants by the ·NO concentration (k' = kobs/[·NO]).
·NO unimolecular dissociation kinetics were measured by ligand displacement reactions. Approximately 1 mg of solid sodium dithionite was added to a 1-cm path length cuvette that was sealed with a rubber septum. The cuvette was flushed with either nitrogen or argon and then completely filled, such that there was no gas space, with anaerobic 0.1 M bis-Tris propane, 0.1 M Cl
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(Eq. 1) |
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RESULTS |
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Effect of Hemoglobin Solutions on Blood Pressure--
The mean
arterial pressure responses to a 50% isovolemic exchange transfusion
with the six different cell-free hemoglobin solutions are shown in Fig.
1. The data are divided into two panels and plotted as the percent change in blood pressure during the base-line and exchange periods. Fig. 1 shows the final 10 min of the
30-min base-line period followed by the 30-min exchange transfusion
period. The x-axis is scaled such that the exchange begins
at time = 0 min. The different types of MAP response observed with
the different hemoglobin solutions used in these experiments can be put
into three categories based on their changes from base line (Table
I): 1) an immediate and sustained
increase (-Hb, Tm-Hb, and o-R-poly-Hb); 2) an
immediate but transient increase (PHP and
82-Hb); and 3) no
significant (p > 0.05) increase (PEG-Hb). The time
period between exposure to cell-free hemoglobin and appearance of a
significant increase in MAP is approximately 8-10 s for the five
groups of rats that show blood pressure increases.
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·NO Reactions at the Heme-- To test the hypothesis that different patterns of MAP response can be explained by different degrees of ·NO scavenging, the reaction of ·NO at the hemes of these six hemoglobins was studied in vitro. Kinetic and equilibrium binding constants were determined for the reaction between ·NO and the reduced, unliganded form of these hemoglobins. The reaction between ·NO and the oxygenated forms of these hemoglobins was also studied.
Bimolecular Association Kinetics--
The bimolecular
recombination time courses for ·NO binding to all six chemically
modified hemoglobins studied, and native human HbA0 as a
control, are identical within experimental error (Table II). Recombination time courses
subsequent to the laser pulse were best described by a single
exponential function. The concentration of free ·NO is in vast
excess of the concentration of photolysed hemes (<1 µM)
and a pseudo-first order approximation is valid for the recombination
reaction. The overall bimolecular association rate constants for these
reactions are obtained by dividing the fitted value of the observed
rate constant by the concentration of ·NO. All of the
hemoglobins studied exhibited values for the association rate constant
that are, within experimental error, equal to 30 µM1 s
1. This value is
essentially the same as the value previously reported for ·NO
binding to native human hemoglobin (25 µM
1
s
1) (21) and indicates that the various types of chemical
modification (i.e. cross-linking, surface modification,
polymerization) have no measurable effect on the room temperature
association kinetics of ·NO binding to the heme of
hemoglobin.
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·NO Oxidation of Oxyhemoglobin--
Bimolecular rate
constants for the reaction of ·NO with the oxygenated forms of
the chemically modified hemoglobins were measured by rapid mixing
techniques. This irreversible reaction proceeds by direct bimolecular
combination of ·NO with O2 bound to hemoglobin and
is not an O2 displacement reaction (18, 19). Time courses
for the ·NO reaction with each of the six chemically modified
hemoglobins studied, and native human HbA0 as a control,
are identical within experimental error (Table II). Overall bimolecular
rate constants for these reactions were obtained by dividing the fitted
value of the observed rate constants by the concentration of ·NO
(kox' = kobs/[·NO], Table II). As observed for
the ·NO bimolecular recombination reaction, there is no
difference in the reaction kinetics between any of the hemoglobins
studied, within experimental noise limits. All hemoglobins studied
yielded values of approximately 30 µM1
s
1 for kox'.
Unimolecular Dissociation Kinetics--
The dissociation of
·NO from each of the hemoglobins was studied by CO replacement
in the presence of excess sodium dithionite. In all cases, the time
courses for the conversion of nitrosylhemoglobin to carbonylhemoglobin
displayed distinctly biphasic kinetic behavior and were fitted to a
double exponential expression (Equation 1) to obtain observed rate
constants. Under the conditions used for these reactions, the values of
the dissociation rate constants were assumed to be equal to the values
of the observed rate constants (Table II). The values of the rate
constants for the slow kinetic phase vary by less than a factor of two,
ranging from 3.6 × 105 s
1 for
82-Hb to 6.1 × 10
5 s
1 for Tm-Hb.
By contrast, the values of the rate constants for the rapid kinetic
phase vary by more than a factor of five, ranging from 19 × 10
5 s
1 for
82-Hb to 104 × 10
5 s
1 for o-R-poly-Hb.
Equilibrium Constants for ·NO Binding--
Due to the
extremely high affinity of hemoglobin binding to ·NO, it is
generally not possible to determine the values of overall dissociation
equilibrium constants by titration methods. In such cases, the
equilibrium dissociation constants are calculated as the ratio of the
respective unimolecular dissociation rate constants to the bimolecular
association rate constants (Kd = k/k', Table II). The values of the equilibrium constants calculated in this
manner vary by a factor of six, ranging from 2.3 pM for 82-Hb to 14 pM for o-R-poly-Hb. The
·NO affinities for these hemoglobins parallel their respective oxygen affinities (see Tables 1 and 2 and Fig.
2). The overall affinity of these
chemically modified hemoglobins for ·NO is principally
controlled by the rate of dissociation from T-state hemoglobin (see
"Discussion"). In the case of
82-Hb and HbA0, both
the small values of the slow kinetic phase rate constants and the
relatively small fractions of fully liganded T-state contribute to the
low value of their overall dissociation rate constants. The hemoglobins
that show an immediate and sustained increase in mean arterial blood
pressure in response to 50% isovolemic exchange transfusion
(
-Hb, Tm-Hb, o-R-poly-Hb, Table I, Fig. 1) exhibit the
weakest ·NO binding affinity (Table II). The hemoglobins that
show either a transient MAP increase (
82-Hb, PHP) or no significant
increase (PEG-Hb) display the tightest ·NO binding.
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DISCUSSION |
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A hypothesis has been advanced by several researchers that
the vasopressor response usually observed upon administration of cell-free hemoglobin solutions is due to ·NO scavenging by
hemoglobin (for a review, see Ref. 5). Physiologic control of vascular
smooth muscle tone is achieved by a dynamic balance of factors that
cause vasoconstriction and vasorelaxation. A reduction in concentration
of EDRF (·NO) in vivo is expected to result in an
increase in vascular smooth muscle tension since a factor believed to
counter vasoconstrictive processes is lost. Furthermore, the time
period between exposure to hemoglobin and appearance of a significant
increase in MAP is approximately 8-10 s for the five groups of rats
that show blood pressure increases. This implies that only a small
amount of cell-free hemoglobin (<10 mg) is required to elicit the
blood pressure responses, and suggests that the rates of the
reaction(s) that cause these responses are very rapid. ·NO binds
to the deoxy-form of hemoglobin with picomolar affinity, and reacts
irreversibly with oxyhemoglobin. Both of these reactions occur with
bimolecular rate constants in excess of 30 µM1 s
1 at 37 °C. Thus it
is easy to assume that cell-free hemoglobin solutions cause
vasoconstriction by rapid removal of EDRF.
Recent evidence in support of this theory comes from experiments that used site-directed mutants of recombinant human hemoglobin designed such that the rates of ·NO reaction with these mutant hemoglobins were reduced relative to that of native hemoglobin (19, 22). The bimolecular rate constants for ·NO-induced oxidation for the resulting mutant oxyhemoglobins were up to 20-fold lower than the rate observed for wild-type Hb-O2. These recombinant hemoglobins elicited vasopressor responses in rats that were significantly diminished relative to that observed with recombinant hemoglobin exhibiting a ·NO reaction rate constant equal to native human hemoglobin. There was a direct correlation between the increase in MAP in vivo and the in vitro rate of ·NO-induced oxidation. These results suggest a causal relationship exists between ·NO reactivity and the vasopressor response, and by modulating the reactivity of hemoglobin toward ·NO, one can reduce the hemoglobin-induced vasopressor effect.
We have studied six different cell-free hemoglobin preparations and
have found three distinct types of MAP response during isovolemic 50%
exchange transfusion in rats (Fig. 1 and Table I): 1) an immediate and
sustained increase (-Hb, Tm-Hb, o-R-poly-Hb); 2) an
immediate and transient increase (
82-Hb, PHP); and 3) no significant
increase (PEG-Hb). Three MAP responses imply that differences in one or
more physical properties of these hemoglobin solutions must exist. If
the differences in MAP response are due to differences in rates of
·NO scavenging, then these hemoglobins are expected to display different ·NO reaction rates and/or affinities that correlate
with the different MAP responses. The value of the rate constant, or
affinity, for the reaction of PEG-Hb with ·NO would be expected
to be less than the corresponding values for any of the other
hemoglobins in this study. The transient MAP increases seen with PHP
and
82-Hb would be explained by intermediate rates or levels of
·NO scavenging, and the large or sustained MAP increases seen
with
-Hb, Tm-Hb, and o-R-poly-Hb would correspond to
the largest rates of reaction, or overall affinities with ·NO.
The results of this study, however, show that differences in MAP
response (Fig. 1) do not correlate with ·NO reaction rates and
inversely correlate with ·NO affinities (Table II, Figs. 1 and
2).
The values of the rate constants determined for the ·NO-induced
oxidation reaction are consistent with those obtained for the bimolecular association reaction with the deoxyhemoglobins. In their
recent study of the mechanism of ·NO-induced oxidation of
hemoglobin, Eich et al. (19) reported bimolecular rate
constant values in the range 30-50 µM1
s
1 for the reaction of ·NO with wild-type human
Hb-O2, which are slightly larger than the values obtained
in this study. We cannot account for the minor discrepancy in these two
studies. However, it is apparent that under identical reaction
conditions, the rate constants for the reaction of ·NO with both
the deoxy- and oxy-forms of all of the chemically modified hemoglobins
examined in this study, and unmodified human hemoglobin, are the
same.
The dissociation rate constants are the only measurable difference in ·NO binding parameters (Table II). The ·NO dissociation time courses are biphasic with unequal amplitudes for the rapid and slow kinetic phases (Table II). This behavior has been reported for native human hemoglobin under conditions that decrease oxygen affinity (i.e. low pH, the presence of inositol hexaphosphate) and have been interpreted as being due to cooperative dissociation of ·NO (16, 23, 24). The kinetic heterogeneity observed in this study suggests cooperative dissociation of ·NO and the existence of significant levels of both high affinity (R) and low affinity (T) Hb-NO conformational states which coexist in equilibrium. As ·NO dissociates from hemoglobin in the presence of CO, a solution of Hb-NO that is partially in the high-affinity (R) state and partially in the low-affinity (T) state is converted to carbonomoxyhemoglobin which is entirely in the high-affinity (R) state.
The observed rate constants for the slow kinetic phase of the
·NO dissociation time courses are approximately the same for all the hemoglobins studied suggesting that there is little difference in
the rates of ·NO dissociation from R-state Hb-NO. Differences in
overall ·NO dissociation rate constants appear to be controlled
by the differences in the rate constant for ·NO dissociation
from T-state Hb-NO. The lowest values for overall ·NO
dissociation rate constants are observed with HbA0 and
82-Hb, and are due to a combination of both a smaller fraction of
T-state Hb-NO and a relatively low value for the rate constant for
·NO dissociation from T state. It is also the case that there is no difference in the association rate constants among the hemoglobins studied. Differences in the overall equilibrium dissociation constants, therefore, are also due to differences in the rate constants for ·NO dissociation from T-state Hb-NO.
The correlation between overall ·NO affinity in vitro
and MAP response in vivo appears to be the opposite of what
is expected, based on the hypothesis that an ·NO scavenging
mechanism accounts for MAP responses and vasoconstriction. Hemoglobins
that are observed to cause the largest and/or most persistent increases
in MAP (-Hb, Tm-Hb, and o-R-poly-Hb) exhibit the
weakest ·NO binding. Hemoglobins that exhibit either transient
or no MAP increase exhibit the highest ·NO affinity. However,
the differences observed upon 50% exchange transfusion must arise from
differences in one or more physical properties exhibited by these
solutions. For example, these hemoglobin solutions display different
O2 binding parameters, molecular weights (Table I) and
different solution properties including viscosity and colloid osmotic
pressure (25, 26).
Because of their size and solution properties, the surface modified hemoglobins (PEG-Hb and PHP) occupy a much greater molecular volume in solution than do cross-linked tetrameric hemoglobins (25). This may be important if the mechanism for cell-free hemoglobin-induced vasoconstriction requires movement of the hemoglobin from the circulation to the extravascular space. It is possible that ·NO scavenging is important only when hemoglobin enters the interstitial space between endothelial cells and smooth muscle cells. If this is the case, then the lack of MAP increase observed with PEG-Hb and the reduced MAP response observed with PHP may be due to molecular size which may prevent passage through endothelial cell junctions.
There is little experimental work that directly assesses the ability of
cell-free hemoglobin to diffuse out of the lumen. Differences in rates
of disappearance of Evans blue dye from the plasma of rats treated with
PEG-Hb or -Hb suggest that greater vascular leaking is induced by
-Hb than by PEG-Hb (7). Fluorescently labeled PEG-Hb has been
shown to extravasate within minutes in the intestine of anesthetized
rats despite observations that suggest that this type of junction is
too small to permit passage of PEG-Hb (27). However, in both these
cases, the time for extravasation appears to be too long to account for
the immediate MAP increases seen in this study. Additionally, it is
unlikely that the difference in MAP response between
82-Hb and the
low O2 affinity tetramers
-Hb and Tm-Hb (Fig. 1) is
due to differences in extravasation since these hemoglobin molecules
are structurally so similar. The differences in MAP response between
these hemoglobin solutions are probably not due to differences in
ability to diffuse out of the lumen. However unlikely, we cannot
discount the possibility that the differences in MAP response are due
to differences in extravascular ·NO scavenging, and further
study of the interaction between cell-free hemoglobin and the
endothelium is required.
It has also been suggested that oxygen-linked S-nitrosylation reactions of hemoglobin may be important in regulation of vascular tone (28, 29). It is possible that different MAP responses may be due to differences in the abilities of chemically modified hemoglobins to participate in these reactions. However, it is not clear whether S-nitrosylation reactions are relevant to extracellular hemoglobin. It also appears that even in the presence of low molecular weight thiols, transnitrosylation reactions occur on time scales that are too slow to account for the immediate MAP increases observed in this study.
Mechanical forces associated with blood flow may play important roles in the control of vascular tone (30). When flow increases, shear stress increases, causing arteries to dilate by endothelial-dependent, nervous-system-independent, relaxation of the smooth muscle cells. For a Newtonian fluid, shear stress is directly proportional to viscosity. In the case of blood (non-Newtonian), or mixtures of blood and cell-free hemoglobin solutions which are Newtonian (26), shear stress increases with increasing viscosity. The effects of solution viscosity on O2 delivery have been observed by comparing tissue oxygenation following hemodilution with either colloids or with crystalloids (31). The differences in MAP responses observed in this study are consistent with this model of shear stress-mediated vasoregulation. PEG-Hb and PHP, which elicit either transient or no MAP increase, exhibit significantly higher viscosities than the other hemoglobin solutions when measured at the same concentration and shear rate (26).
We find a direct correlation between ·NO and O2 affinities (Fig. 2). Interestingly, it has been suggested that the presence of a low oxygen affinity cell-free hemoglobin solution in vivo may not necessarily lead to a corresponding high level of O2 delivery to the tissues (32-34). Cell-free hemoglobin may overcome a diffusive limitation to O2 delivery (35) resulting in abnormally high O2 levels in the regulatory arterioles. This has the potential to produce an autoregulatory response in which vasoconstriction causes increased vascular resistance and reduced flow. In this model, the shape and position of the O2 equilibrium binding curve, the total O2 carrying capacity, and the fluid viscosity are crucial in determining the amount of O2 delivered to the regulatory regions of the microcirculation (6).
The differences in MAP response observed in this study are consistent
with the hypothesis that MAP responses are, in part, dictated by the
O2 binding properties. A comparison of the intramolecularly cross-linked hemoglobin tetramers illustrates the effect of
O2 binding properties. Solutions of Tm-Hb and -Hb
both produce an immediate and sustained increase in MAP (Table I, Fig.
1). These hemoglobins are structurally very similar, exhibit identical solution properties, at the same concentration (25, 26), and similar
O2 affinity and cooperativity (Table I). The physical properties of
82-Hb are also nearly identical to those of Tm-Hb and
-Hb, with the exception of the O2 binding properties.
82-Hb binds O2 with higher affinity and less
cooperativity than either Tm-Hb or
-Hb (Table I). Solutions of
82-Hb elicit a diminished and transient MAP response relative to
Tm-Hb and
-Hb. Thus we find a direct inverse correlation between
O2 affinity and vasopressor response. The hemoglobins that
cause sustained MAP increases exhibit the highest
p50 values (lowest O2 affinity)
whereas the hemoglobins that cause either transient or no MAP increase
exhibit the lowest p50 values (highest
O2 affinity, Table I and Fig. 2).
Physiological control of vascular smooth muscle tone relies on many factors, any or all of which may be affected by the presence of a cell-free hemoglobin. This study has addressed only one such factor: the ability of a cell-free hemoglobin to react with ·NO at the heme ligand binding site, and has found no correlation between ·NO reactivity and MAP response. It is more likely that the O2 affinities and solution properties of hemoglobin solutions are more important in determining the vasopressor effect. The full physiological relevance of ·NO scavenging reactions will only be discovered through continued investigations of the mechanisms involved in regulating blood pressure.
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ACKNOWLEDGEMENTS |
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We thank the following companies for their generous gifts of hemoglobin solutions used in this study: Hemosol, Inc., Etobicoke, Ontario, Canada; Enzon, Inc., Piscataway, NJ; and Apex Biosciences, Inc., Research Triangle Park, NC.
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FOOTNOTES |
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* This work was supported in part by United States Public Health Service, National Institutes of Health NHLBI Grant HL48018.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.
§ To whom correspondence should be addressed: Veterans Affairs Medical Center, 3350 La Jolla Village Dr. (111-E), San Diego, CA 92161. Tel.: 619-552-8585 (ext. 7299); Fax: 619-552-7578; E-mail: rrohlfs{at}ucsd.edu.
1
The abbreviations used are: ·NO, nitrogen
monoxide; bis-Tris, 1,3-bis[tris(hydroxymethyl)methylamino]; EDRF,
endothelium-derived relaxing factor; MAP, mean arterial pressure; PE,
polyethylene; POE, polyoxyethylene; -Hb, human hemoglobin
cross-linked with bis-(3,5-dibromosalicyl)fumarate between the lysine
99 residues of the
subunits;
82-Hb, human hemoglobin reacted
with trimesoyl tris(methyl phosphate) to make a two-point
intramolecular cross-link between the
subunits at lysine 82;
HbA0, purified native human hemoglobin;
o-R-poly-Hb, human hemoglobin polymerized with ring-opened raffinose; PEG-Hb, bovine hemoglobin surface conjugated to
methoxypolyoxyethylene glycol; PHP, human hemoglobin modified by
covalent attachment of pyridoxal-5'-phosphate and surface conjugated to
-carboxymethyl,
-carboxy- methoxypolyoxyethylene; Tm-Hb,
human hemoglobin reacted with trimesoyl tris(methyl phosphate) to make
a three-point intramolecular cross-link between the
subunits at
lysine 82 and valine 1 of one
chain.
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