OXIDATIVE MECHANISMS OF HEMOGLOBIN-BASED BLOOD SUBSTITUTES^*

Abstract

Chemically or genetically altered cell-free hemoglobin (Hb) has been developed as an oxygen carrying therapeutic. Site-directed modifications are introduced and serve to stabilize the protein molecules in a tetrameric and/or a polymeric functional form. Direct cytotoxic effects associated with cell-free Hb have been ascribed to redox reactions (involving either 1 or 2 electron steps) between the heme group and peroxides. These interactions are the basis of the pseudoperoxidase activity of Hb and can be cytotoxic when reactive species are formed at relatively high concentrations during inflammation and typically lead to cell death. Peroxides relevant to biological systems include hydrogen peroxide (H₂O₂), lipid hydroperoxides (LOOH), and peroxynitrite (ONOO⁻). Reactions between Hb and peroxides form the ferryl oxidation state of the protein, analogous to compounds I and II formed in the catalytic cycle of many peroxidase enzymes. This higher oxidation state of the protein is a potent oxidant capable of promoting oxidative damage to most classes of biological molecules. Further complications are thought to arise through the disruption of key signaling pathways resulting from alteration of or destruction of important physiological mediators.

• Hemoglobin
• Blood substitutes
• Oxidative stress

HEMOGLOBIN-BASED BLOOD SUBSTITUTES AND THE VASCULAR SYSTEM

Several potential problems can be created when stroma-free Hb (SFH) is present in plasma and/or in close proximity to the vascular system as recent animal and human studies have shown [[1]]. The vascular endothelium has emerged as the primary target of Hb-based toxicity due to its proximity to the circulating protein. Hb can thus reach nitric oxide (NO) and potentially disturb the physiological balance between NO and the powerful biological “peroxides” (i.e. peroxynitrite (ONOO⁻) and hydrogen peroxide (H₂O₂)) (Figure 1). The interactions between cell-free Hb with oxidants may potentially be detrimental to both Hb and the vasculature [[1]]. Recent animal studies and calculation of NO kinetics at the vessel wall showed that under normal physiological conditions a red blood cell-free zone is created that reduces the consumption of vascular NO by RBCs due to normal intravasular flow [[2]]. In addition, The unstirred layer of plasma around the RBC and its membranes present additional hurdles for NO to reach intraerthyrocytic Hb. The enzymatic machinery within red blood cells would normally reduce any ferricHb formed from the normal autoxidation process or the oxidation of Hb by NO. In contrast RBCs a cell-free Hb which lack these natural protective mechanisms will undoubtedly be close enough to the sources of NO which presumably react with NO resulting in vasoconstriction/hypertension [[1]].

Figure 1. Hb-based blood substitutes and the vascular system. (A) Under normal physiological conditions, NO acts as a vasodilator and antioxidant. There is more NO than O₂^·− (superoxide is kept at remarkably low levels by a high concentration of the enzyme superoxide dismutase) thus, any pro-oxidant effects of ONOO⁻ and HOOH are suppressed by the anti-oxidant function of NO. (B) Cell-free Hb induces vasocostriction due to the scavenging of NO. In addition, the balance is disrupted, thus O₂^·− level exceeds NO formation resulting in the loss of the beneficial effects of NO and the concomitant formation of ONOO⁻ and HOOH. The interactions between Hb and these oxidants may thus be detrimental to both Hb and the vasculature (adapted from reference [[1]]).

Reactive oxygen and nitrogen species have been traditionally regarded as toxic byproducts of aerobic metabolism. The biological peroxides discussed herein have been implicated as regulators of redox sensitive cell signaling pathways [[3]]. For example, reactive species have been implicated in the regulation of hematopoiesis [[4]]. Several studies have shown that H₂O₂ regulates transcriptional and translational events in many cell types [5-6]. The exact targets that H₂O₂ reacts with, to either stimulate or repress a given pathway is not known, but downstream targets include the mitogen activated protein (MAP) kinases, nuclear factor kappa B (NFκB) and hypoxia inducible factor (HIF-1). These are important components of numerous redox sensitive-signaling pathways that link extracellular stimuli to gene regulation [[7]]. Similarly, lipid peroxides are intermediates in the cascade of reactions forming compounds that mediate an inflammatory response [[8]]. Recently more specific roles have been reported including modulation of endothelial cell NO synthase [[9]]. Relatively little is known of the signaling roles of ONOO⁻ and in fact its production is generally considered a deleterious event. However, recent data indicate that this is not always the case and at low physiological concentrations (in the nM range) ONOO⁻ may have cardioprotective functions. Furthermore, endogenous production of this reactive nitrogen species (RNS) in endothelial cells modulates shear dependent activation of JNK [[10]]. These effects are not deleterious, and in fact, protect the cell from subsequent oxidative or nitrosative insults.

The effects of reactions between Hb and biologically relevant peroxides in the context of cell signaling have not been explored. The concentrations of SFH that can be achieved upon administration are high (in the mM range in terms of heme) [[11]] and can potentially compete with endogenous reactions that consumes the peroxides. No systematic studies have been conducted addressing these issues, but reactions between Hb and H₂O₂ generated during reperfusion of ischemic endothelial cells were demonstrated [[12]]. Therefore, similar to NO, the effects of Hb on cell function may be more subtle than oxidative damage mediated by HbFe⁴⁺ and involve perturbation of redox sensitive signaling pathways (Figure 2).

Figure 2. Potential modulation of cell signaling by Hb-based blood substitutes. Oxidative stress and/or shear stress stimulates production of NO and O₂^{· −} in endothelial cells, which react together to form ONOO⁻. Scavenging of NO by oxyHb is predicted to increase H₂O₂ production from O₂^·−, a process catalyzed by superoxide dismutase (SOD). OxyHb can be oxidized to metHb by NO, ONOO⁻, and H₂O₂ (not shown for purposes of clarity). In turn metHb can modulate ONOO⁻ and H₂O₂ concentrations by reactions that lead to ferryl Hb (Hb⁴⁺=O) production. Both reactive oxygen and nitrogen species can regulate transcription and translation processes. The downstream targets of reactive species remain poorly defined. Examples include regulation of sGC, JNK, NFκB, iron response element (IRE) activity and hypoxia-inducible factor (HIF-1). Examples of specific genes known to be regulated by reactive species includes the antioxidants heme oxygenase (HO-1) and glutathione, and the pro-inflammatory adhesion molecules VCAM-1 and ICAM-1 (adapted from reference [[17]]).

MECHANISM OF PEROXIDE INTERACTIONS WITH Hb

The reactions between the respiratory proteins Hb or myoglobin (Mb) and H₂O₂ have been extensively characterized and proceed via a two electron oxidation process (for review see 11). Reaction of H₂O₂ with either the ferrous (Fe²⁺) or ferric (Fe³⁺) Hb produces the ferryl species, a potent oxidant in which the heme is formally two oxidation equivalents above the Fe²⁺ and is referred to as Fe⁴⁺ [[13]]. If reactions occur with the Fe³⁺ form, protein-based radicals are also formed [[14]]. The radical represents the second oxidizing equivalent, and both peroxyl and carbon-based radicals are formed simultaneously. A cyclic mechanism including peroxide dependent oxidation of Fe³⁺ heme and reduction of Fe⁴⁺ heme and radical has been proposed [[15]]. Hb-based radical species have been detected in whole blood indicating that H₂O₂ can react with HbFe⁴⁺/HbFe³⁺ in red blood cells in the presence of endogenous mechanisms which remove H₂O₂ [[16]]. HbFe⁴⁺ is a potent oxidant capable of oxidatively damaging most biological substrates including lipids, nucleic acids and amino acids. Some of the biological examples of Hb/Mb redox reactions have been recently reviewed and are summarized in table 1 [[17]]. Formation of Fe⁴⁺ of both Hb and Mb was correlated to cytotoxicity in an endothelial cell culture model of ischemia-reperfusion and more so in cells that lack their anti-oxidative mechanisms such as glutathione [[12]], [[18]]. Recent insights have identified the redox cycle between MbFe³⁺ and MbFe⁴⁺ as an important modulator of injury in kidneys of rhabdomylotic rats [[19]]. This added more impetus to research efforts by many groups investigating the effects of oxidative reactions mediated by hemoproteins in other pathological processes (see table 1). A further example includes oxidative damage to LDL mediated by Hb, a process that has been correlated with free Hb concentrations in hemodialysis patients [[20]]. Oxidative damage to apoB (the protein component of LDL) is a key event that transforms LDL into a pro-atherogenic form that in turn promotes atherogenesis.

Table 1. Biological Examples of Hb/Mb Redox Reactions

Ischemia/reperfusion

Brain Injury

Blast Injury

Rhabdomyolysis

Atherosclerosis

Blood Substitutes (?)

The globin-based radical of HbFe⁴⁺ has recently been detected in normal human blood by EPR [[21]]. Interestingly, the source of H₂O₂ in blood is considered to be the dismuation of superoxide anion radical (O₂^·−) produced via the autoxidation of Hb. In addition to intermolecular cross-links, globin radical formation also forms intramolecular cross-links between the heme and amino acids [[14]]. The functional consequences of these modifications are not yet known, but are likely to lead to protein degradation and release of iron. In the case of Mb, the treatment with H₂O₂ leads to covalent alteration of the prosthetic group with concomitant formation of a protein-bound adduct that transforms Mb from an oxygen storage protein to an oxidase, capable of producing additional H₂O₂ [22-23]. Visible absorption, fluorescence and low temperature EPR studies recently showed that the reaction of oxyHb with H₂O₂ leads to heme degradation [[24]]. This process involves the reaction of an additional H₂O₂ molecule with HbFe⁴⁺ to produce HbFe³⁺ and O₂^·− radical anion. Superoxide produced in the heme pocket reacts with the porphyrin entity resulting in heme degradation and release of iron [[24]]. Interestingly, the autoxidation of Hb has recently been shown to proceeds by the same mechanism, i.e. the ferric/ferryl cycle [[25]].

DIASPIRIN CROSS-LINKED Hb, A MODEL CASE FOR THE STUDY OF OXIDATIVE MECHANISMS

Diaspirin cross-linked Hb (DBBF-Hb), an intramolecularly cross-linked tetramer with bis(3,5-dibromosalicyl)fumarate (DBBF), has been extensively studied in vitro and in animal models [[26]]. In recent years, considerable research and development efforts have been invested in the commercial analogue, DCLHb or HemAssist™, produced by Baxter Healthcare Inc. and its non-commercial analogue, DBBF-Hb, produced by the U.S. Army. The production and development of both Hbs have recently been halted by both the Army and Baxter due to, in the case of DCLHb, reports of excessive fatalities in late clinical trials [27-28]. In vitro studies on this Hb revealed that besides modifying ligand interactions, α-subunits cross-linking with DBBF can also affect the tendency of this Hb to undergo oxidative modification and the production of HbFe⁴⁺ in solution [[12]], [[29]]. The reaction with H₂O₂ produces persistent DBBF-HbFe⁴⁺ in solution suggesting that it lacks an effective pseudoperoxidase activity [[15]]. The differences in the extent and nature of the radical formed in the cross-linked Hb may have important implications in that the amount of oxidative damage it can induce.

In vitro evidence on the detection of the of DBBF-HbF⁴⁺ has recently been documented in a number of experimental settings; monolayer of endothelial cells, endothelial cells subjected to ischemia/reperfusion and in cells that lack the antioxidant mechanism i.e., glutathione [[12]], [[18]], [[29]]. DBBF-Hb may have favorable oxygen binding properties, however, the unique redox chemistry, i.e. suppressed ability to remove H₂O₂ and susceptibility to oxidative damage, may be a contributory factor in its toxicity.

Cell-free Hb may present a low risk to healthy individuals with normal redox status, however, patients with a compromised vasculature and poor antioxidant status, i.e., diabetes, hypertension, myocardial infraction and acute ischemic stroke, may be at greater risk, as recent clinical trial failures with DCLHb have demonstrated [27-28]. Recent revelation by Baxter based on some hundred preclinical studies in several small and large animals on the development of DCLHb induced myocardial lesions is of interest in this regard [[30]]. These lesions were characterized by mild to moderate focal-to-multifocal myocardial degeneration and/or necrosis in a highly vascularized portion of the myocardium. Neither serum enzyme activities nor ECG analysis and echocardiography were able to predict the incidences of these lesions. Interestingly, the infusion of Hb solutions with reduced affinity to react with NO resulted in significant decreases in the incidence and severity of these lesions [[30]]. Although the primary event responsible for the microvascular effects of Hb solutions appears to be the removal of NO by Hb, subsequent oxidative reactions between Hb and oxidants of the vascular system (i.e. H₂O₂ and ONOO⁻) may have potentially lead to a vascular inflammatory cascade of reactions progressing to multi-organ failure (Figure 2).

PROTECTION STRATEGIES: INHIBITION OF THE REACTIONS BETWEEN Hb AND PEROXIDES

Several antioxidant enzymes and molecules safely detoxify peroxides both in the intra and extracellular compartments [[31]]. Despite the presence of these systems, formation of HbFe⁴⁺ in whole blood has been demonstrated [[21]]. However, HbFe⁴⁺ does not accumulate and oxidative damage to the red blood cell is likely prevented due to a comproportionation reaction between HbFe⁴⁺ and HbFe²⁺ [[32]]. The product of this reaction is HbFe³⁺. Coupled with the endogenous metHb reductase system these reactions represent a protective pathway that can remove peroxide formation without exposing the red blood cell to an oxidative stress [[32]].

The role of this protective pathway in cell-free Hb solutions is less clear due to the lower concentrations of HbFe²⁺ and the decreased capacity of plasma to reduce HbFe⁴⁺ back to HbFe³⁺. However, biological reductants present in the plasma may limit oxidative reactions mediated by HbFe⁴⁺ formed outside the erythrocyte [[31]]. Therapeutically, Hb-based oxygen carriers that do not readily react with peroxides or show decreased HbFe⁴⁺ reactivity would be good candidates for a blood substitute. Structural modification of Hb can modulate HbFe⁴⁺ reactivity. For example cross linking of the β-subunits rather than the α-subunits produced proteins that have better pseudoperoxidase activity [[15]]. Several genetically modified myoglobins and Hbs were shown to exhibit anti-ferryl and anti NO properties when the heme environment is re-engineered to prevent the interaction of peroxide and NO with heme group [33-34]. Surface modification of pyridoxal phosphate cross-linked human Hb with polyoxyethylene (PHP) produced less HbFe⁴⁺ than non-conjugated human Hb [[35]]. Interestingly, later published work by the same group revealed that PHP was contaminated with red cell antioxidant enzymes, i.e. SOD and catalase [[36]]. Another very promising strategy is to crosslink Hb with trace amounts of SOD and catalase [[37]]. This strategy prevents HbFe⁴⁺ production and protects against oxidative damage mediated by this Hb derivative. Similar results have been obtained using polynitroxylated Hb that was shown to inhibit ROS dependent activation of leukocyte adherence to the endothelium [[38]]. The conjugation of Trolox (a water-soluble vitamin E analog) and a known anti-ferryl reagent to O-raffinose-polymerized Hb appeared to provide 96% antioxidant protection in vitro to this Hb-based oxygen therapeutics [[39]]. Cross-linking of Hb with adenosine and GSH, a surface modifier, also provided pharmacological and antioxidant characteristics to bovine Hb as was recently shown [[40]].

Table 2. Antioxidant Protective Strategies

Chemical and/or Genetic engineering of Hb

Cross-linking of RBC antioxidative enzymes to Hb

Cross-linking of nitroxide to Hb

Cross-linking of Trolox (vitamin E analogue) to Hb

Stroma-free Hb (SFH) containing endogenous antioxidative RBC's enzymes

Cross-linking Hb with adenosine and GSH