Robert M. Winslow

Arterial blood pressure responses to cell-free hemoglobin solutions and the reaction with nitric oxide

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.

Microvascular and tissue oxygen distribution

Understanding of oxygen delivery by the microcirculation has been dominated by the unitary component analysis of Krogh and Erlangen focussed on oxygen transport mediated by single capillaries, oxygenation of tissue as a whole being extrapolated from findings on oxygen exchange in these vessels. This analysis is under revision since capillaries are not sole sources of oxygen. It is increasingly apparent that arterioles are a significant equivalent source, while venules may serve as sinks for capillary and arteriolar oxygen. As a consequence detailed descriptions of the architecture of the microcirculation based on the tissue cylinder conceptualization does not yield new information given the non-exclusive role of capillaries as purveyors of oxygen to tissue. In the present study we investigate how tissue is oxygenated directly from the arteriolar supply on the basis of current results with newly developed optical techniques for the measurement of local intra- and extravascular pO2 by phosphorescence decay. This methodology shows that tissue regions between arterioles and venules have essentially uniform tissue pO2. The only experimentally detectable gradients in pO2 are those present in the immediate vicinity of arterioles. Findings on vascular longitudinal gradients are used to devise a model that links convective and diffusive processes, showing how blood viscosity, blood oxygen-carrying capacity and the slope of the oxygen dissociation curve are linked in determining intravascular and tissue pO2. The integrated approach provides a numerical basis for interpreting consequences of alterations in transport properties of blood applicable to the field of blood substitutes.

Heme Oxygenase-1–Derived Carbon Monoxide Contributes to the Suppression of Acute Hypertensive Responses In Vivo

The enzyme heme oxygenase, which exists in inducible (HO-1) and constitutive (HO-2) isoforms, catalyzes the degradation of heme to biliverdin and CO in mammalian tissues. CO has been implicated in the control of vascular tone in a manner similar to that for NO. In the present study, we investigated the contribution of the heme oxygenase/CO pathway to the modulation of acute hypertensive responses in vivo induced by (1) alphaalphaHb, a chemically modified hemoglobin known to scavenge NO, and (2) NG-nitro-L-arginine methyl ester (L-NAME), a competitive NOS inhibitor. Experiments were carried out in conscious rats in which femoral arteries and veins were surgically catheterized 1 or 5 days before treatment with the vasoconstrictor agents. Intravenous infusion of alphaalphaHb (8% solution) or L-NAME (30 micromol/kg) [corrected] produced an acute and significant increase in mean arterial pressure (P<0.05) in rats at 5 days after catheter implantation. In contrast, no change in blood pressure was observed when alphaalphaHb or L-NAME was infused 1 day after the surgical intervention. The suppression of the hypertensive response observed at 1 day after surgery correlated with a significant (P<0.05) HO-1 expression in aorta, heart, and liver as well as increased aortic CO production and cGMP levels. At 1 day after surgery, pretreatment of animals with the heme oxygenase inhibitor zinc protoporphyrin IX (50 micromol/kg IP) markedly decreased aortic CO and cGMP levels and completely restored the vasoconstrictor effects of both alphaalphaHb and L-NAME. These results provide evidence for a crucial role of the heme oxygenase/CO pathway in the regulation of blood pressure under stress conditions in vivo.

MP4, a new nonvasoactive PEG-Hb conjugate

BACKGROUND: Vasoconstriction has been an obstacle to clinical development of Hb‐based O2 carriers. It is proposed that this limitation can be overcome by increasing molecular size and oxygen affinity.

Progress in the development of RBC substitutes.

Volume 41, February 2001 TRANSFUSION 287 www.transfusion.org Considerable progress has been made in the development of RBC substitutes in the last decade, with several preparations advancing from the preclinical stage of testing to Phase III clinical trials. Although the primary focus has been on the development of a pharmacologic substitute that could carry and deliver oxygen (O2) in the place of banked blood, the unique properties of the RBC substitutes under study have led to the exploration of applications beyond the conventional use of RBCs in transfusions. The term “RBC substitute,” although widely used (including in medical literature databases), does not convey the diverse properties of, and potential clinical applications for, these materials. The availability of O2-carrying therapeutic agents for clinical use could have a significant impact on the practice of blood banking and transfusion medicine. This review describes the various substitutes under development and the progress that has been made in clinical trials, and considers some of the potential implications of their use. Several recent comprehensive reviews provide additional details regarding the scientific progress in developing these new products.1-6 RBCs have several unique properties. First, they contain a very high concentration of Hb and protect it from degradation, which would be rapid if the Hb were free in the plasma. Second, since Hb is contained within the cell, it does not come in direct contact with tissues, where it might otherwise release its heme and damage the tissue. Third, the intraerythrocytic environment prevents oxidation of Hb to methemoglobin, which is not capable of transporting O2 and may promote injurious oxidative reactions. 7

Resuscitation with polyethylene glycol-modified human hemoglobin improves microcirculatory blood flow and tissue oxygenation after hemorrhagic shock in awake hamsters

OBJECTIVE To determine whether resuscitation with polyethylene glycol-modified human hemoglobin (MalPEG-Hb), an oxygen-carrying blood replacement fluid with 4 g/dL Hb, viscosity of 2.5 cP, colloid osmotic pressure of 49 mm Hg, and p50 of 5.5 mm Hg, improves systemic and microvascular variables after hemorrhage compared with shed blood (SB) and 5% hydroxyethyl starch (HES). SETTING Laboratory. SUBJECTS Golden Syrian hamsters. DESIGN Prospective study. INTERVENTIONS Hamsters implemented with a skin fold chamber were hemorrhaged 50% of blood volume and resuscitated with 50% shed blood volume (SB, HES, or MalPEG-Hb). MEASUREMENTS AND MAIN RESULTS Shock and resuscitation were monitored for 1 hr each. Microvascular events were characterized in terms of vessel diameter, flow velocity, functional capillary density, and Po(2) in arterioles, venules, and extravascular tissue. Systemic variables include mean arterial pressure, heart rate, Po(2), Pco(2), pH, and base excess. MalPEG-Hb resuscitation increased functional capillary density to 64% vs. 44% for SB and 32% for HES relative to baseline before shock. Microvascular flow increased 16% for MalPEG-Hb relative to baseline and remained decreased by 44% for SB and 80% for HES. Hemoglobin concentration was 10.4 g/dL with SB, 7.5 (6.8 g/dL in red blood cells and 0.9 g/dL in plasma) with MalPEG-Hb, and 7.5 g/dL with HES, leading to tissue Po(2) of 19, 8, and 5 mm Hg respectively. Calculations of oxygen extraction show that 0.9 g/dL of MalPEG-Hb increased oxygen extraction per gram of red cell hemoglobin in the tissue analyzed compared with SB. These measurements correlate well with a systemic indicator of recovery, base excess, 5.4 +/- 4.7 (MalPEG-Hb), 1.7 +/- 3.8 (SB), and -0.3 +/- 5.7 (HES). CONCLUSION The presence of 0.9 g/dL of high oxygen affinity MalPEG-Hb improves microvascular blood flow and oxygen transport during shock to a significantly greater extent than that attainable with blood or HES.

Colloid osmotic properties of modified hemoglobins: Chemically cross-linked versus polyethylene glycol surface-conjugated

Colloid osmotic pressures of hemoglobin solutions containing unmodified, intramolecularly cross-linked, intermolecularly polymerized, or polyethylene glycol (PEG) surface-conjugated hemoglobin have been measured to determine their macromolecular solution properties. Tetrameric and polymeric hemoglobins show nearly ideal solution behavior: whereas, hemoglobins conjugated to PEG have significantly higher colloid osmotic activity and exhibit solution non-ideality. From these studies, the average calculated molecular weights are 65.300 +/- 3500 for unmodified and intramolecularly cross-linked hemoglobin tetramers, 156,000 for ring-opened raffinose polymerized human hemoglobin, 97,000 for pyridoxalated human hemoglobin conjugated to a carboxy-PEG polymer, and 117,000 for bovine hemoglobin conjugated to a methoxy-PEG polymer. The calculated radius of gyration for tetrameric hemoglobins is 2.9 +/- 0.2 nm compared to 4.9 nm for the polymerized hemoglobin, and 7.2 and 14.1 nm for the human and bovine PEG-conjugated hemoglobins, respectively. Exclusion volumes are calculated to be 823 +/- 148 nm3 for tetramers, 4000 nm3 for polymers, and 13,000 nm3 and 94,000 nm3 for human and bovine PEG-conjugated hemoglobins, respectively. These studies show that polyethylene glycol conjugated to surface amino groups greatly increases the effective macromolecular size of hemoglobin in solution.

A multicenter clinical study of the safety and activity of maleimide-polyethylene glycol-modified hemoglobin (hemospan®) in patients undergoing major orthopedic surgery

Background:Hemospan® (Sangart Inc., San Diego, CA), a polyethylene glycol–modified hemoglobin with unique oxygen transport properties, has successfully completed a phase I trial in healthy volunteers. Because adverse events are expected to increase with age, the authors conducted a phase II safety study of Hemospan in elderly patients undergoing elective hip arthroplasty during spinal anesthesia. Methods:Ninety male and female patients, American Society of Anesthesiologists physical status I–III, aged 50–89 yr, in six Swedish academic hospitals were randomly assigned to receive either 250 or 500 ml Hemospan or Ringer’s acetate (30 patients/group) before induction of spinal anesthesia. Safety assessment included vital signs and Holter monitoring from infusion to 24 h, evaluation of laboratory values, and fluid balance. The hypothesis to be tested was that the incidence of adverse events would be no more frequent in patients who received Hemospan compared with standard of care (Ringer’s acetate). Results:Three serious adverse events were noted, none of which was deemed related to study treatment. Liver enzymes, amylase, and lipase increased transiently in patients in all three groups. There were no significant differences in electrocardiogram or Holter parameters, but there was a suggestion of more bradycardic events in the treated groups. Hypotension was less frequent in the treated patients compared with controls. Conclusions:In comparison with Ringer’s acetate, Hemospan mildly elevates hepatic enzymes and lipase and is associated with less hypotension and more bradycardic events. The absence of a high frequency of serious adverse events suggests that further clinical trials should be undertaken.

Acute changes in systemic blood pressure and urine output of conscious rats following exchange transfusion with diaspirin-crosslinked hemoglobin solution.

This report describes acute changes in systemic blood pressure and urine output observed after a 50‐percent isovolemic exchange transfusion (ET) with diaspirin‐crosslinked hemoglobin (alpha alpha Hb). Stroma‐free Hb was crosslinked between the alpha chains by using a 14C‐labeled diaspirin, bis(3,5‐dibromosalicyl)fumarate. Forty conscious, chronically cannulated rats underwent ET with 14C‐labeled alpha alpha Hb solution (8.0 g/dL [80 g/L]). This resulted in systemic hypertension for 3 to 4 hours after ET (mean arterial pressure rose from 120 to 145 torr at 1 to 2 hours after ET) and mild bradycardia for 2 to 3 hours (heart rate decreased from 420 to 335 beats/min [bpm] before stabilizing at 360 +/− 10 bpm). This was accompanied by significant diuresis immediately after ET (5‐ 6‐fold increase in urine output, which normalized after 12 hours), and mild hemoglobinuria. The total amount of Hb recovered in the urine was < 5 percent of the injected dose. Reversed‐phase high‐performance liquid chromatography and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis confirmed the presence of crosslinked alpha alpha Hb molecules in the urine. Renal excretion of radioactivity was significantly greater, with 20 percent of total radioactivity being eliminated within 24 hours. The plasma half‐life for alpha alpha Hb was 5 hours (administered dose, 2.4 g Hb/kg body weight). Thus, infusion of alpha alpha Hb caused a transient systemic hypertension, and intramolecular crosslinking alone was not enough to exclude completely the filtration of alpha alpha Hb by the kidneys.

Pilot-scale preparation of hemoglobin solutions.

Publisher Summary Cell-free hemoglobin, suitably modified to confer a physiological oxygen affinity and prolonged plasma retention, is a candidate for a temporary substitute for red blood cells. Such a solution would probably find wide clinical acceptance because the risks of disease transmission from banked human blood are significant. This chapter discusses the pilot-scale preparation of hemoglobin solutions. The production facility is housed in four separate, but connected, rooms: one contains the system for generation of pyrogen-free water (PFW), the second is used for reagent preparation, the third contains the filtration assemblies and the bioreactor, and the fourth is a 4° cold room that houses the preparative high-performance liquid chromatography (HPLC) equipment. An additional large walk-in cold room is available to store large tanks of buffers. The apparatus used for hemoglobin preparation consists of several components, including a manifold for emptying bags of outdated blood; cross-flow filtration devices for washing, lysis, and purification; a bioreactor for cross-linking hemoglobin; a preparative-scale HPLC apparatus; and a hood for final filling of product into sterile bags.