Arterial Blood Pressure Responses to Cell-free Hemoglobin Solutions and the Reaction with Nitric Oxide*

Ronald J. RohlfsDagger §, Eric Bruner, Albert ChiuDagger , Armando GonzalesDagger , Maria L. GonzalesDagger , Douglas Magde, Michael D. Magde Jr.Dagger , Kim D. VandegriffDagger , and Robert M. WinslowDagger

From the Dagger  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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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 µM-1 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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha  subunits (alpha alpha -Hb). Isovolemic exchange transfusions in rats with a solution of alpha alpha -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 alpha alpha -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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha  chains at lysine 99 (alpha alpha -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 beta  chains at lysine 82 (beta 82-Hb, 6.8 g/dl, 4.3 mM heme) or a three-point cross-link between the beta  chains at lysine 82 and valine 1 of one beta  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 alpha -carboxymethyl, omega -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.

Nitrosylhemoglobin (Hb-NO) samples were prepared by equilibrating anaerobic solutions of deoxyhemoglobin under 1 atm of ·NO gas. A Sephadex G-25 column (1 cm × 20 cm) was equilibrated with anaerobic 0.1 M bis-Tris propane, 0.1 M Cl-, pH 7.4 buffer. A few crystals of solid sodium dithionite were added to 0.5 ml of stock oxyhemoglobin and immediately passed through the column. The deoxyhemoglobin was eluted off the column through a needle directly into a tonometer containing 1 atm of ·NO gas, prepared as described above, and equilibrated to produce a [·NO] = 1.97 mM Hb-NO solution. This solution was used directly for dissociation experiments or diluted with appropriate volumes of anaerobic and ·NO-equilibrated buffers to produce Hb-NO samples at a heme concentration of 100 µM, and a defined ·NO concentration, for laser photolysis experiments.

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-, pH 7.4, buffer that had been equilibrated previously with 1 atm of carbon monoxide gas ([CO] = 1 mM). The ·NO displacement reaction was initiated by introducing a few microliters of stock nitrosylhemoglobin solution prepared as described above. The excess dithionite reacts with free ·NO dissolved in solution but does not react with ·NO bound to hemoglobin (16). As the ·NO dissociates, it is destroyed by the dithionite, and CO binds to the heme. Kinetic transients were monitored as the conversion of Hb-NO to Hb-CO with a Milton Roy 3000 diode array spectrophotometer. Time courses were fitted to a biexponential expression (Equation 1) using an iterative nonlinear least squares algorithm to obtain the observed rate constants and the fractional amplitudes of each kinetic phase. In Equation 1, At is the absorbance at a given point in the time course, Delta Atot is the fitted total absorbance change, frapid is the fitted fractional amplitude of the rapid kinetic phase, Ainfinity is the fitted final absorbance, and krapid and kslow are the fitted rate constants for the rapid and slow kinetic phases, respectively. Under these conditions,
A<SUB>t</SUB>=f<SUB><UP>rapid</UP></SUB>&Dgr;A<SUB><UP>tot</UP></SUB><UP>exp</UP>(<UP>−</UP>k<SUB><UP>rapid</UP></SUB>t)+(1−f<SUB><UP>rapid</UP></SUB>)&Dgr;A<SUB><UP>tot</UP></SUB><UP>exp</UP>(<UP>−</UP>k<SUB><UP>slow</UP></SUB>t)+A∞ (Eq. 1)
the observed rate constants are virtually identical to the actual dissociation rate constants (17). An overall dissociation rate constant (k) for each hemoglobin was calculated as the mean of the dissociation rate constants for the rapid and slow kinetic phases weighted by the fractional contributions (k = (frapidkrapid + (1 - frapid)kslow). Overall equilibrium constants were calculated as the ratio of the overall dissociation rate constant to the corresponding association rate constant (Kd = k/k').

The kinetics of ·NO-induced oxidation of Hb-O2 were measured by conventional rapid mixing techniques using a stopped-flow spectrophotometer (Applied Photophysics model 17MV, London, UK). Solutions of air-equilibrated hemoglobin were mixed against anaerobic solutions of ·NO, and the conversion of oxyhemoglobin to methemoglobin was monitored by the absorbance change at 419 nm. The value of the bimolecular rate constant for this reaction is reported to be extremely large, on the order of 10 µM-1 s-1 (18, 19), so the concentration of ·NO after mixing was kept low (<= 10 µM) in order to minimize loss of the reaction time course in the mixing time of the stopped-flow apparatus. Under these conditions, the rate of the autoxidation reaction of ·NO with dissolved O2 is negligible compared with the rate of reaction with oxyhemoglobin (20). The concentration of Hb-O2 after mixing was at least a factor of 5 less than that of ·NO (usually [Hb-O2] = 0.5 µM) in order to use a pseudo first order approximation. The low Hb-O2 concentrations resulted in small absorbance changes, and ten kinetic transients were signal-averaged for each time course in order to improve the signal-to-noise ratio. The time courses were fitted to a single exponential expression using an iterative non-linear least squares algorithm to obtain the observed rate constants. The bimolecular rate constants were obtained by dividing the observed rate constants by the ·NO concentration (kox' = kobs/[·NO]).

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 (alpha alpha -Hb, Tm-Hb, and o-R-poly-Hb); 2) an immediate but transient increase (PHP and beta 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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Fig. 1.   Arterial blood pressure response to 50% isovolemic exchange transfusion with cell-free hemoglobin solutions. The percent change in mean arterial pressure is plotted versus time with the x-axis scaled such that the last 10 min of the 30-min base-line period are shown, and the exchange transfusion begins at time = 0 min. The symbols represent the average value at a given time point for each group of animals. The error bars represent the standard error of the mean. Results for the six groups are divided into two panels. A, MAP response for animals receiving alpha alpha -Hb (open circle , n = 6), PHP (black-triangle, n = 16), and PEG-Hb (square , n = 5). B, MAP response for animals receiving Tm-Hb (bullet , n = 9), o-R-poly-Hb (black-square, n = 5), and beta 82-Hb (triangle , n = 4). Data for alpha alpha -Hb and PEG-Hb are taken from Migita et al. (7).

                              
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Table I
Properties of modified hemoglobins
Types of hemoglobin modifications are described in Winslow (4). Molecular mass values for the hemoglobin cross-linked tetramers (alpha alpha -Hb, Tm-Hb, and beta 82-Hb) are not significantly different than unmodified human hemoglobin. Molecular mass values for PHP, PEG-Hb, and o-R-poly-Hb were taken from Vandegriff et al. (25). Oxygen equilibrium binding parameters (p50, n50) were measured in 0.1 M bis-Tris propane, 0.1 M Cl-, pH 7.4, 37 °C by the method described in Vandegriff et al. (13). The errors represent the standard error of the mean. Entries in the column labeled "Vasopressor Effect" are based on statistical analyses of the data shown in Fig. 1. MAP responses that increased and remained significantly above base line (p < 0.05) throughout the exchange transfusion period are considered "Sustained." MAP values that initially increase significantly above base line, then fall to values not significantly different from baseline during the exchange transfusion are considered "Transient." MAP responses not significantly different from baseline are labeled as "None" since no vasopressor effect is observed.

The average MAP of rats receiving solutions of alpha alpha -Hb (n = 6) increased from 110 ± 2 to 135 ± 3 mm Hg and achieved the maximal value 5-6 min after start of the exchange (Fig. 1A). A similar result was observed with rats that received Tm-Hb (n = 9, Fig. 1B). The MAP increased from 115 ± 3 mm Hg to a maximal value of 135 ± 4 mm Hg, the maximum also occurring 5-6 min after the start of exchange. Rats that received o-R-poly-Hb (n = 5) showed a smaller average sustained MAP increase, rising from 115 ± 3 to 126 ± 2 mm Hg and achieved a maximal MAP value more rapidly (2-3 min) than alpha alpha -Hb or Tm-Hb rats (Fig. 1B). In all three of these cases, the rapid increase in MAP coupled with a maximal value that is sustained throughout the exchange transfusion period leads to the "square wave" appearance of these time courses.

Transient increases in the average MAP were observed in rats that received solutions of either PHP or beta 82-Hb. For both of these Hb solutions, the rapid increase was followed by a decrease in MAP value back to base-line levels before conclusion of the 30-min exchange period. In the case of rats receiving PHP, the MAP increased at the start of the exchange from a base-line value of 112 ± 2 mm Hg to a maximal value of 126 ± 2 mm Hg at 3 min into the exchange (n = 16, Fig. 1A). The MAP then decreased to 118 ± 4 mm Hg at 15 min after the start of exchange, a value not significantly different from base line. The beta 82-Hb group showed an increase similar in magnitude to that of the PHP group (from 108 ± 4 to 123 ± 4 mm Hg in 3 min) with a more rapid return to base line, 10 min into the exchange (n = 4, Fig. 1B).

The MAP response in rats to 50% isovolemic exchange transfusion with PEG-Hb is different from the responses observed in the five other groups (n = 5, Fig. 1A) (7). No significant increase in the average MAP above the base-line value (109 ± 4 mm Hg) was observed for the PEG-Hb group at anytime during exchange transfusion. A small increase in MAP (maximal value of 114 ± 4 mm Hg) was observed approximately 3 min after the start of exchange transfusion, and this trend returned to base line within 10 min after the start of the exchange.

·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 µM-1 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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Table II
Nitric oxide reaction parameters
Each parameter listed for each hemoglobin represents the mean of at least three separate determinations, all determined in 0.1 M bis-Tris propane, 0.1 M Cl-, pH 7.4, 23 °C. The errors represent the standard error of the mean. The bimolecular association rate constants (k') were measured by flash photolysis of nitrosylhemoglobin samples prepared in the presence of 1.97 mM (1 atm) ·NO and 0.197 mM (0.1 atm) ·NO. Dissociation rate constants were determined by ligand displacement with CO in the presence of excess sodium dithionite ([dithionite] = 1-2 mM). Rate constants (krapid, kslow) and amplitudes (frapid, %) for the rapid and slow kinetic phases of the displacement reaction were obtained from biexponential fits of the time courses (Equation 1) (fraction of the slow kinetic phase equals 1 - fraction rapid). The overall dissociation rate constants (k) represent the weighted mean of both kinetic phases based on the fractional amplitudes for each kinetic phase. Equilibrium constants (Kd) were calculated as the ratio of the dissociation rate constant to the association rate constant (Kd = k/k'). Bimolecular rate constants for the reaction of ·NO with oxyhemoglobin were determined by stopped-flow rapid mixing.

·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 µM-1 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 × 10-5 s-1 for beta 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 beta 82-Hb to 104 × 10-5 s-1 for o-R-poly-Hb.

The amplitudes for the rapid and slow kinetic phases for a given Hb-NO dissociation time course are not equal, indicating that the observed biphasic kinetic behavior cannot be due simply to differences between the alpha  and beta  chains. The rapid kinetic phase accounts for approximately 35% of the total absorbance change observed for the dissociation of ·NO from alpha alpha -Hb, Tm-Hb, PHP, and o-R-poly-Hb. For beta 82-Hb, the rapid kinetic phase accounts for 21% of the total absorbance change and, for HbA0, the rapid phase accounts for 25% of the total absorbance change. PEG-Hb is markedly different from the other hemoglobins studied in that the rapid kinetic phase accounts for a higher percentage of the overall ·NO dissociation time course, 54%. Since each time course yields two observed dissociation rate constants, due to significant populations of both R- and T-state nitrosylhemoglobins (see "Discussion"), overall dissociation rate constants were estimated by taking the average values for the observed rate constants for each kinetic phase, weighted by their respective contributions to the total absorbance change (Table II).

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 beta 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 beta 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 (alpha alpha -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 (beta 82-Hb, PHP) or no significant increase (PEG-Hb) display the tightest ·NO binding.


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Fig. 2.   Correlation between ·NO and O2 affinities. Equilibrium dissociation constants for ·NO binding to chemically modified hemoglobins (Kd)are plotted versus the corresponding partial pressures of oxygen at half saturation (p50). The error bars represent the standard error of the mean. Values are taken from Tables I and II. Cell-free hemoglobins fall into two groups: 1) those that elicited an immediate and sustained MAP increase (alpha alpha -Hb, Tm-Hb, and o-R-poly-Hb) (bullet ), and 2) hemoglobins that caused either a transient (PHP and beta 82-Hb) or no significant MAP increase (*PEG-Hb) (triangle ). The numbers in parentheses are the values (mm Hg) for the maximum increase in MAP over base line during exchange transfusion (Fig. 1). The symbols are within a 95% confidence level of the fitted linear regression (solid line).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 µM-1 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 (alpha alpha -Hb, Tm-Hb, o-R-poly-Hb); 2) an immediate and transient increase (beta 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 beta 82-Hb would be explained by intermediate rates or levels of ·NO scavenging, and the large or sustained MAP increases seen with alpha alpha -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 µM-1 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 beta 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 (alpha alpha -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 alpha alpha -Hb suggest that greater vascular leaking is induced by alpha alpha -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 beta 82-Hb and the low O2 affinity tetramers alpha alpha -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 alpha alpha -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 beta 82-Hb are also nearly identical to those of Tm-Hb and alpha alpha -Hb, with the exception of the O2 binding properties. beta 82-Hb binds O2 with higher affinity and less cooperativity than either Tm-Hb or alpha alpha -Hb (Table I). Solutions of beta 82-Hb elicit a diminished and transient MAP response relative to Tm-Hb and alpha alpha -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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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; alpha alpha -Hb, human hemoglobin cross-linked with bis-(3,5-dibromosalicyl)fumarate between the lysine 99 residues of the alpha  subunits; beta 82-Hb, human hemoglobin reacted with trimesoyl tris(methyl phosphate) to make a two-point intramolecular cross-link between the beta  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 alpha -carboxymethyl, omega -carboxy- methoxypolyoxyethylene; Tm-Hb, human hemoglobin reacted with trimesoyl tris(methyl phosphate) to make a three-point intramolecular cross-link between the beta  subunits at lysine 82 and valine 1 of one beta  chain.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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