Problems with Hemoglobinbased Blood Substitutes in the Microcirculation

Arteriolar Constriction

Role of Hemoglobin as a Nitric Oxide Scavenger

Many experimental studies have reported an increase in blood pressure after administration of hemoglobin solutions, and pilot clinical trials have confirmed this observation. One theory to explain this phenomenon is that arterioles constrict due to removal of the endogenous vasodilator, nitric oxide (NO), by the hemoglobin. This theory is supported by studies showing that administration of the NO precursor, L-argi-nine, at the same time as DCLHb reduces the vasopressor effects. It is thought that when Hb is enclosed in red blood cells its ability to scavenge NO from the endothelium is diminished because of the existence of a cell-free plasma layer in the microvessels. The vasopressor effects of Hb-based blood substitutes are generally considered to be a disadvantage because vasoconstriction will reduce the blood supply to the peripheral circulation. However, in some cases the accompanying increase in blood pressure could be advantageous. For example, hemoglobin solutions may improve recovery from cardiac arrest by simultaneously increasing coronary perfusion and oxygenation. In addition, in cases of sepsis when an increase in inducible NO syn-thase activation causes an overproduction of NO, leading to hypotension, a scavenger of NO would be beneficial. Apart from scavenging the vasodilator, NO, modified hemoglobins may also cause the endothelium to release the vasoconstrictor, endothelin, or activate endothelin receptors.

Role of Hemoglobin as an Oxygen Deliverer

Recently, a third explanation has been proffered to explain hemoglobin-induced vasoconstriction: That is, the Hb provides excess oxygen to tissue, and in response, the arterioles narrow to reduce blood flow and hence tissue oxygen delivery. This response is known as "autoregulation." For this reason, a new blood substitute, Hemospan (Sangart, San Diego, CA), has been designed to reduce oversupply of oxygen. Hemospan is prepared by conjugating polyethylene glycol to hemoglobin tetramer from outdated human blood [3]. This product, in contrast to most of the other blood substitutes, has a high oxygen affinity (low po2) and a high viscosity (similar to blood rather than to plasma). The high

Cell-free hemoglobin

Conjugated tetramer

Conjugated tetramer

oxygen affinity ensures that the modified Hb will release oxygen only to tissue that is anoxic, and thus autoregulatory vasoconstriction, produced by excess oxygen delivery, will be eliminated. It is thought that because of the higher viscosity of this product, the shear stresses in the microvessels will be maintained, thus ensuring that the endothelium continues to release NO at a rate similar to that obtained with blood. However, this is unlikely to occur because shear stress is proportional not only to viscosity, but also to the rate of strain in the vessel (dv/dr, where v = flow velocity and r = radius). A higher viscosity will reduce the volumetric flow rate, and hence flow velocity and rate of strain. Thus the shear stress will not increase with increased viscosity unless the flow rate is maintained. At present Hemospan is in the preclinical phase of testing, but Phase I trials are planned.


It is important to remember that blood substitutes should not just be considered as oxygen carriers. One other important property of whole blood is that it has a high antioxidant capacity and makes a significant contribution to the total antioxidant defenses of the body. Infusion of resuscitation fluids may therefore influence the antioxidant capacity of plasma, dependent on their composition. It is vital that the antioxidant capacity of a transfusion fluid be adequate because reperfusion of the circulation after loss of blood triggers production of reactive oxygen species (ROS). The effects of dilution of blood on its antioxidant properties were demonstrated elegantly by Moison et al. [4]. They found that addition of isotonic saline, or pasteurized plasma protein solution (lacking uric acid and vitamin C with reduced sulfhydryl level), to cord blood from babies decreased the peroxyl radical trapping capacity. In contrast, fresh, frozen plasma did not lower this capacity. It was hypothesized that the use of resuscitation fluids with low antioxidant capacity may temporarily decrease the ability of the baby to inactivate reactive oxygen species.

However, not only do most modified hemoglobins have a low antioxidant capacity; there is evidence that modified hemoglobins, injected in vivo, produce highly reactive oxygen species themselves, leading to tissue damage [5]. The ROS are formed because Hb is susceptible to oxidation and auto-oxidation. Some Hb-based blood substitutes have been shown to oxidize more readily than Hb contained in red blood cells in response to chemical modifications aimed at lowering oxygen affinity. Although cell-free Hb may present a low risk to people with normal redox status, patients who are sick and have a poor antioxidant supply may be at risk. Oxidative damage is particularly dangerous in the microcirculation because gaps form between the endothelial cells resulting in excess leakage of plasma components into the interstitium. Such leakage disturbs the fluid balance between blood and tissue and alters the kinetics of delivery of intravascularly injected drugs and endogenous enzymes and hormones to various tissues.

Possible Mechanisms Responsible for Hemoglobin-Induced Toxicity

Three possible mechanisms can account for the propensity of modified Hbs to form oxidative by-products. First, the ferrous and ferric forms of Hb can react with hydrogen peroxide (H2O2), produced by neutrophils or macrophages, to form the highly reactive ferryl intermediate Hb Fe4+, and this may lead to heme degradation and cytotoxicity. A second possible redox mechanism involves the release of free iron from Hb after oxidative damage. The free iron then catalyses the reaction between H2O2 and superoxide (O"^2), both produced by phagocytes, to form the hydroxyl radical (Off) by the Fenton reaction. Finally, NO can react with oxygen free radicals, or with Hb, to produce more ROS.

Ferryl hemoglobin. It has been postulated that the excess H2O2 reacts with ferrous Hb (HbFe2+) and ferric, or "met" Hb (HbFe3+), in vivo, to initiate further oxidation cycles resulting in the formation of highly reactive ferryl-Hb (HbFe4+). Specifically, when HbFe2+ reacts with H2O2, it donates two electrons to the H2O2, to form HbFe4+ and OH-. In HbFe4+ the iron center is at a higher oxidation state. Despite its transient nature, Fe4+ heme can peroxidize lipids, degrade carbohydrates, and modify proteins. The further interaction of HbFe4+ with H2O2 results in the formation of rhombic heme, which is considered to be one of the best measures of the toxicity of a blood substitute. The rhombic heme in which the geometry of the iron is distorted due, in some instances, to the chemical modification of the protein, then initiates a cascade of oxidative side reactions resulting in the formation of free iron.

Fenton reaction. In the hemoglobin molecule the hemes are normally bound in pockets from which water is largely excluded. Conformational changes that open up the heme pockets and allow greater access of water and small anions favor the conversion of the heme iron to the ferric or "met" state. The reaction (auto-oxidation), which also produces superoxide anions, occurs spontaneously in modified hemoglobins. A key study showed that 72 hours after infusion of glutaraldehyde polymerized bovine Hb into an animal, almost 40 percent of circulating hemoglobin is in the met form [6]. This is important because it has been estimated that met Hb concentrations greater than 10 percent significantly decreases the ability of Hb to deliver oxygen to tissues. The met Hb then reacts with H2O2 to release free iron and free heme, which decomposes H2O2 to form hydroxyl radical (Off) and hydroxide anion (OH-) by the Fenton reaction:

The Fe3+ that is formed can react with superoxide radicals to produce more Fe2+:

This Fe2+ then undergoes the Fenton reaction to produce more Off. Thus, the combination of H2O2 and Fe2+ represents a highly toxic potential that contributes to peroxidation in hemoglobin-containing systems. One Off can result in the conversion of many hundred fatty acid side chains into lipid hydroperoxides. Iron is also capable of catalyzing the production of alkoxy and peroxy radicals from lipid peroxides, and the production of these reactive species could contribute to tissue injury.

In normal blood, the reservoir of heme iron is compartmentalized within the erythrocyte, which limits its ability to act as a catalyst. Erythrocytes are rich in antioxidant enzymes, such as superoxide dismutase (SOD) and cata-lase, which inactivate the O"*2 and H2O2, respectively. These enzymes are in close proximity to Hb within the ery-throcytes, and although Hb can release catalytic iron when exposed to oxidant stress, injury is limited because the enzymes react with the resultant ROS before they reach the cell membrane.

Reactions involving nitric oxide. Nitric oxide, which is constitutively produced by endothelial cells and is produced by macrophages when they are activated, can have either a protective or a deleterious effect on tissue in the presence of Hb. The deleterious effect can arise in two ways. One way is from its interaction with O"*2 to form peroxynitrite anion, ONOO-:

The amount of O2- present determines the amount of ONOO- that is formed. The concentration of O2- in the circulation is kept extremely low by a high concentration of superoxide dismutase (SOD). A balance between NO^ and O2- is therefore maintained under physiological conditions, and the reaction between the two to form ONOO- is limited. When the circulation is perfused with a modified Hb, the Hb reacts very quickly with the NO^ and the resulting reduction in NO^ drives the formation of ONOO-. On protonation, ONOO- decomposes to the highly reactive OH\ Nitric oxide can also react with oxyHb (Hb(Fe-O2)) to produce met Hb, which is unstable and easily releases iron that catalyzes production of OH\ The protective effect of NO^ occurs when it scavenges the damaging oxidant peroxynitrite:

It is obvious from these reactions that the role played by NO\ whether it acts as a pro-oxidant or as an antioxidant, depends on the relative amounts of NO^ and (ROS) that are present. The fact that Hb has a high affinity for NO^ has the potential for upsetting an existing balance between NO^ and ROS.

Effects of Hemoglobin-based Blood Substitutes on Microvascular Permeability

It is well known that excess ROS oxidize lipids of the cell membranes. Lipid peroxidation damage of membrane components is thought to play an important role in increasing microvascular permeability. When hemoglobin-based blood substitutes are injected into the circulation, excess ROS, such as H2O2, O2-, Off, and Fe4+, will form if the hemoglobin increases its oxidation state. Since H2O2 can easily diffuse across cell membranes, and O2- can traverse membranes via the chloride anion channel, it is likely that these (ROS) will leave the microvasculature and gain access to other cells in the tissue. This action can have deleterious effects. For example, if the ROS reach mast cells in the interstitium they will cause the mast cells to degranulate and release a selection of inflammatory mediators, including histamine, which will increase microvascular permeability. In addition, degranulating mast cells release eosinophil and neutrophil activating factors, triggering these leukocytes to release more ROS. Surprisingly, only a few studies have been performed to determine whether injection of a hemoglobin-based blood substitute into the circulation significantly increases the accumulation of ROS in surrounding tissue. In one study [5], a bolus injection of DBBF-Hb into the circulation of rats was found to rapidly produce excess ROS, as detected by fluorescence of dihydrorhodamine-123, in the intestinal mucosa.

Although it is known that formation of the unstable form of hemoglobin, met Hb, proceeds rapidly after injection of modified hemoglobins into the circulation, and that

Figure 2 Image pair of FITC-albumin (A) and rhodamine phalloidin-stained (B) 10-minute DBBF-Hb preparation showing venules. Note small leaks (arrowhead in A) and extensive leak (arrow in A). The small leaks (A) are coincident with the endothelial junctions as identified by rhodamine-stained peripheral actin rims (arrowhead in B). Precise positions of the small leaks coincide with discontinuities in PARs. Scale bars = 25 mm. (From Ref. [7]).

Figure 2 Image pair of FITC-albumin (A) and rhodamine phalloidin-stained (B) 10-minute DBBF-Hb preparation showing venules. Note small leaks (arrowhead in A) and extensive leak (arrow in A). The small leaks (A) are coincident with the endothelial junctions as identified by rhodamine-stained peripheral actin rims (arrowhead in B). Precise positions of the small leaks coincide with discontinuities in PARs. Scale bars = 25 mm. (From Ref. [7]).

ROS increase microvascular permeability, very few studies have been performed to determine whether injection of hemoglobin-based blood substitutes increase microvascular permeability. One study [7] showed that bolus injection of DBBF-Hb, in rats, increased venular leakage to fluores-cently labeled bovine serum albumin (FITC-BSA) and produced mast cell degranulation in the rat mesentery. Such changes are characteristic of an inflammatory response. Focal leakage of FITC-BSA from mesenteric venules following a 10-minute perfusion with DBBF-Hb is illustrated by a figure from this study (Figure 2). The upper panel demonstrates focal leaks of FITC-BSA. Small leaks (arrowhead) and a large leak (arrow) are visible. The lower panel shows the same segment of venule, but stained for F-actin. The arrowhead indicates that small leaks are sometimes coincident with focal breaks in the peripheral endothelial actin rim. This type of focal macromolecular leakage is similar to that observed in the presence of histamine. The discrete nature of the leakage means that it is not easily detected in studies in which only the average fluorescence in the tissue is measured. It is disadvantageous for a potential blood substitute to cause microvascular leakage, because the substitute itself will rapidly leave the circulation, and in addition, alterations in transvascular exchange of plasma proteins will disturb the fluid balance between blood and tissue. Increased microvascular leakage also changes the kinetics of delivery of intravascularly injected drugs, and of endogenous enzymes and hormones, to various tissues. When transfusions are needed, for example after hemor-rhagic shock, it is important that regulation of microvascu-lar exchange not be compromised.

Methods to Reduce Tissue Damage by Blood Substitutes in Vivo

The diagram in Figure 3 depicts the various mechanisms by which modified hemoglobins can cause tissue damage. From this diagram, it is clear that there are at least three different types of agents that could be administered to reduce microvascular damage: (a) antioxidants, (b) iron chelators, and (c) mast cell stabilizers. Very little progress has been made using iron chelators and mast cell stabilizers, and so only antioxidants will be considered.

Use of superoxide dismutase and catalase. Superoxide dismutase and catalase are endogenous antioxidants for O2'" and H2O2, respectively. Several methods currently incorporate SOD and catalase into cross-linked Hb (i.e., Ref. [8]). This technique ensures that the free-radical scavengers are in close contact with the source of the ROS, the hemoglobin, and also effectively addresses the problem that SOD has a short half-life in blood (10 to 40 minutes). An alternative way of increasing the half-lives of SOD and catalase is to bind them to PEG. Polyethylene glycol-SOD has a half-life of several days, and PEG linkage increases the half-life of catalase from 2 to 50 hours.

Use of nitroxides. Another antioxidant that has been investigated for use with blood substitutes is nitroxide. Nitroxides are able to scavenge O2'", and thus can act as

Possible Mechanisms by Which Modified

Hemoglobins Can Cause Microvascular Damage

Possible Mechanisms by Which Modified

Hemoglobins Can Cause Microvascular Damage

Free Iron / Fsnton Reaction

Figure 3 Diagram to show possible mechanisms by which modified hemoglobins can cause microvascular damage. Hb(Fe-O2): oxyhemoglobin; NO': nitric oxide; HbFe2+: ferrous hemoglobin; met Hb: ferric hemoglobin; H2O2: hydrogen peroxide; Hb(Fe4+=O): oxyferryl hemoglobin; OH: hydroxyl radical.

potent antioxidants. However, free nitroxide is cleared very rapidly from the circulation and very little is left even 5 minutes after intravenous infusion. For this reason a polynitroxylated hemoglobin-based oxygen carrier has been developed in which nitroxide molecules are cova-lently bound to Hb so that the circulatory half-life of the nitroxide molecules is greatly increased [9]. At this time there is no published record of the ability of hemoglobins, conjugated with antioxidants, to minimize oxidative tissue damage.

Use of selenium. Recently, the protective effect of selenium on Hb-mediated lipid peroxidation has been investigated for use with blood substitutes. The rationale for using selenium is that it is a very powerful antioxidant. Selenium is thought to act as an antioxidant in the body because it is a component of the enzyme, glutathione peroxidase, which catalyzes removal of H2O2. Hemoglobin's oxidative reactions are very complex, and so total protection cannot be achieved using hydroxyl radical scavengers exclusively. Sodium selenite (Na2SeO3), when administered orally or intravenously, has been shown to significantly reduce the microvascular leakage associated with bolus injection of DBBF-Hb in rats. In addition, Na2SeO3 reduces the oxidation rate of DBBF-Hb while in the presence of oxidants in

Free Iron / Fsnton Reaction

vitro. Thus it appears that Na2SeO3 moderates hemoglobin-induced damage at least partly through its interactions with the hemoglobin molecule itself, and that there is no need for glutathione peroxidase to be involved in the process. However, the disadvantage of this treatment is that selenium compounds might only be useful adjuncts to Hb-based blood substitutes that can be reduced by interaction with selenium.

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