Serial oxygen equilibrium and kinetic measurements during RBC storage.

OBJECTIVES: To contribute to the understanding of the biochemical changes associated with the RBC storage lesion. AIM: To investigate changes in O(2) equilibrium and on/off kinetic rates during routine cold storage. BACKGROUND: As RBCs are stored between 1 and 6°C numerous biochemical changes occur within the RBCs, including changes in the properties of the haemoglobin itself. This study serially analysed for the first time the O(2) equilibrium and on/off kinetic rates across the RBC membrane during routine storage. METHODS/MATERIALS: The oxygen binding (k(on) ) and offloading (k(off) ) constants were measured in fresh RBCs and then in AS-5-preserved RBCs at weekly intervals, along with oxygen equilibrium curves (OECs), 2,3-Diphosphoglycerate (2,3-DPG), p50 and the Hill number (n). RESULTS: The k(on) increased slightly as the 2,3-DPG and p50 decreased during storage, whereas the k(off) remained largely unchanged. The OECs demonstrated the expected increase in O(2) affinity, whereas the Hill number was unchanged during storage. CONCLUSION: In spite of the biochemical, structural and functional changes associated with the storage of RBCs, their in vitro interactions with oxygen were largely preserved through 42 days of storage.

Redox side reactions of haemoglobin and cell signalling mechanisms.

Cell-free chemically modified or recombinant haemoglobins developed as oxygen therapeutics are designed to correct oxygen deficit caused by ischaemia in a variety of clinical settings. Oxidative processes, which are in some cases enhanced when modifications are introduced that lower oxygen affinity, can limit the safety of these proteins. Direct cytotoxic effects associated with haemoglobins have been ascribed to the redox reactions between haemoglobin and biological peroxides [i.e. hydrogen peroxide (H2O2), lipid peroxides (LOOH) and peroxynitrite (ONOO-)]. Biochemical changes at the cellular, tissue and organ levels have been documented to occur in response to haemoglobin oxidative reactions. These peroxides have been implicated as regulators of redox sensitive cell signalling pathways. The effects of reactions between haemoglobin and biologically relevant peroxides may be more subtle than oxidative damage and may thus involve perturbation of redox sensitive signalling pathways. In this review, a brief outline of the role of cell-free haemoglobin in oxidative and cell-signalling pathways and the implications of these reactions on the safety and efficacy evaluation of haemoglobin-based oxygen carries are presented.

Redox cycling of diaspirin cross-linked hemoglobin induces G2/M arrest and apoptosis in cultured endothelial cells.

It is hypothesized that oxidative reactions of hemoglobin driven by reactive oxygen species in the vasculature lead to endothelial cell injury or death. Bovine aortic endothelial cells were incubated with diaspirin cross-linked hemoglobin (DBBF-Hb), developed as a hemoglobin-based oxygen carrier, and hydrogen peroxide (H(2)O(2)), generated by the glucose oxidase system. The low steady flux of H(2)O(2) oxidizes the ferrous form of DBBF-Hb and drives the redox cycling of ferric and ferryl DBBF-Hb. Cells underwent rounding, swelling and detachment, and accumulated in the G2/M phase of the cell cycle. G2/M arrest preceded the onset of apoptosis as determined by increases in phosphatidylserine (PS) externalization and sub-G1 events. Redox cycling of unmodified hemoglobin also led to G2/M arrest and apoptosis. The rate and extent of DBBF-Hb oxidation correlated with the onset and extent of G2/M arrest and apoptosis and induced significant decreases in soluble reduced thiols. Earlier depletion of glutathione by pretreatment with buthionine sulfoximine rendered cells more susceptible to G2/M arrest and apoptosis. The caspase inhibitor, z-VAD-fmk, had no effect on the induction of G2/M arrest but completely inhibited the subsequent increases in PS externalization and sub-G1 events. Catalase inhibited DBBF-Hb oxidation, the loss of thiols, and the onset of G2/M arrest and apoptosis. These data support a causative role for the ferric-ferryl redox cycle in the development of endothelial cell injury.

Effects of glutaraldehyde polymerization on oxygen transport and redox properties of bovine hemoglobin.

Crosslinking of bovine Hb (HbBv) with glutaraldehyde produces a mixture of low oxygen affinity (P(50)) tetrameric and polymeric Hb species (PolyHbBv). Under physiological conditions the P(50) of HbBv and PolyHbBv were 27 and 35 mmHg, respectively. The dependence of the P(50) on pH and chloride ions and the cooperativity (n(50)) of the protein were diminished as a result of glutaraldehyde modification. Rapid kinetic studies showed greater overall rates of oxygen dissociation (k(off)) with little or no change in the association of CO (k(on)) to the modified protein. The rate of nitric oxide (NO)-induced oxidation of the PolyHbBv was slightly lower than that of HbBv. Autoxidation rate of PolyHbBv was 1.4 times faster than that of HbBv. The reaction of hydrogen peroxide (H2O2) with the ferrous (Fe(2+)) and ferric (Fe(3+)) forms of the proteins led to the formation of a more stable ferrylHb (Fe(4+)) in the case of PolyHbBv. Glutaraldehyde polymerization of HbBv alters its normal allosteric mechanisms, autoxidation kinetics and other related redox properties, which may compromise its function and cause greater toxicity when used as an oxygen transport fluid.

Redox reactions of hemoglobin and myoglobin: biological and toxicological implications.

Direct cytotoxic effects associated with hemoglobin (Hb) or myoglobin (Mb) have been ascribed to redox reactions (involving either one- or two-electron steps) between the heme group and peroxides. These interactions are the basis of the pseudoperoxidase activity of these hemoproteins 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, lipid hydroperoxides, and peroxynitrite. Reactions between Hb/Mb 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. Free iron, released from Hb, also has the potential to promote oxidative damage via classical "Fenton" chemistry. It has become increasingly evident that Hb/Mb redox reactions or their by-products play a critical role in the pathophysiology of some disease states. This review briefly discusses the reactions of Hb/Mb with biological peroxides, potential cytotoxicity and the impact of these interactions on modulation of cell signaling pathways regulated by these reactive species. Also discussed in this article is the role of heme-protein chemistry in relation to the toxicity of hemoproteins.

Biological action of nitric oxide donor compounds on platelets from patients with sickle cell disease.

Several lines of evidence point to the potential role of nitric oxide (NO) in the pathophysiology, as well as in the therapy, of sickle cell disease (SCD). In this study, we compared the effects of NO on platelets from normal individuals and from patients with SCD. Three NO donors were used to deliver NO to platelets: sodium 2-(N, N-diethylamino)-diazenolate-2-oxide (DEANO), S-nitrosocysteine (CysNO) and sodium trioxdintrate (OXINO or Angeli's salt). ADP-induced platelet aggregation, CD62P expression, PAC-1 binding and calcium elevation were evaluated in paired studies of normal and SCD subjects. DEANO significantly reduced aggregation in SCD platelets compared with normal platelets. DEANO similarly reduced the extent of CD62P expression in SCD platelets. All NO donors reduced PAC-1 binding, but there were no significant differences between platelets from normal or SCD subjects. Calcium elevation, as induced by ADP, was not altered by the presence of NO donors. However, when platelets were stimulated with thrombin, there was an increased initial response of SCD platelets compared with normal platelets. Taken together, these data suggest that the mode of NO delivery to platelets may produce various physiological responses and the optimization of NO delivery may contribute to reducing platelet aggregation in sickle cell disease.

Oxidative mechanisms of hemoglobin-based blood substitutes.

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-based blood substitutes and the hazards of blood radicals.

Cell-free hemoglobins, chemically altered or genetically expressed in microbial host systems, have been developed as oxygen-carrying therapeutics. Site-directed modifications are introduced and serve to stabilize the protein molecules in a tetrameric and/or a polymeric functional form. Animal studies, as well as recent clinical studies, have suggested these proteins probably deliver oxygen to tissues. However, concerns still persist regarding the interference of hemoglobin and its oxidation products with the vascular redox balance, potentially impeding its clinical usefulness. This article reviews our current understanding of heme-mediated toxicities and some of the emerging protective strategies used to overcome hemoglobin side reactions.

Interactions of hemoglobin with hydrogen peroxide alters thiol levels and course of endothelial cell death.

We investigated cellular injury and death induced by ultrapure human Hb (HbA(0)) and its diaspirin cross-linked derivative DBBF-Hb in normal and glutathione (GSH)-depleted bovine aortic endothelial cells subjected to hydrogen peroxide (H(2)O(2)). HbA(0) underwent extensive degradation and heme loss, whereas DBBF-Hb persisted longer in its ferryl (Fe(4+)) form. The formation of ferryl HbA(0) or ferryl DBBF-Hb was associated with a significant decrease in endothelial cell GSH compared with the addition of H(2)O(2) or Hbs alone. This effect was inhibited by catalase, but not by superoxide dismutase or deferoxamine mesylate. The presence of HbA(0) and DBBF-Hb reduced H(2)O(2)-induced apoptosis, as measured by cell morphology, annexin V binding assay, and caspase inhibition, consistent with the ability to consume H(2)O(2) in an enzyme-like fashion. However, the pattern of cell death and injury produced by HbA(0) and DBBF-Hb appeared to be distinctly different among proteins as well as among cells with and without GSH. These findings may have important implications for the use of cell-free Hb as oxygen therapeutics in patients with coexisting pathologies who may lack antioxidant protective mechanisms.

Effect of nitric oxide and nitric oxide donors on red blood cell oxygen transport.

A mechanism has been proposed in which nitric oxide (NO) may bind to cysteine beta93 and be transported by haemoglobin from the lungs to the tissues and modify vascular tone. In addition, it has been reported that treatment of sickle cell anaemia blood with 80 p.p.m. NO gas in air shifts the oxygen affinity, as measured by P50 to the left. We exposed normal and sickle cell anaemia blood to 80 p.p.m. NO in air for 1 h in vitro and found no change in P50 of either normal or sickle cell blood. In addition, we exposed normal and sickle cell blood in buffer to aqueous NO (NO gas dissolved in buffer) at varying concentrations and found that the induced left shift in P50 correlates strongly and linearly with methaemoglobin formation. We also treated normal and sickle cell blood with other nitric oxide donors, such as sodium 2-(N, N-diethylamino)-diazenolate-2-oxide (DEANO), S-nitrosocysteine (CysNO) and sodium trioxodinitrate (OXINO, or Angeli's salt). In all cases, we found a dose-dependent increase in methaemoglobin that was strongly correlated with the dose-dependent P50 reduction. Our data do not support the report that low NO concentrations can selectively increase the oxygen affinity of sickle cell blood without affecting methaemoglobin levels significantly. NO, however, may have benefit in sickle cell disease by other mechanisms.

Nitric oxide-mediated heme oxidation and selective beta-globin nitrosation of hemoglobin from normal and sickle erythrocytes.

Nitric oxide (NO) has been reported to modulate the oxygen affinity of blood from sickle cell patients (SS), but not that of normal adult blood (AA), with little or no heme oxidation. However, we had found that the NO donor compounds 2-(N, N-diethylamino)-diazenolate-2-oxide (DEANO) and S-nitrosocysteine (CysNO) caused increased oxygen affinity of red cells from both AA and SS individuals and also caused significant methemoglobin (metHb) formation. Rapid kinetic experiments in which HbA(0), AA, or SS erythrocytes were mixed with CysNO or DEANO showed biphasic time courses indicative of initial heme oxidation followed by reductive heme nitrosylation, respectively. Hemolysates treated with CysNO showed by electrospray mass spectrometry a peak corresponding to a 29 mass unit increase (consistent with NO binding) of both the beta(A) and beta(S) chains but not of the alpha chains. Therapeutic use of NO in sickle cell disease may ultimately require further optimization of these competing reactions, i.e., heme reactivity (nitrosylation or oxidation) versus direct S-nitrosation of hemoglobin on the beta-globin.

Site-specific modifications and toxicity of blood substitutes. The case of diaspirin cross-linked hemoglobin.

Safe and effective hemoglobin-based blood substitutes may be advantageous over conventional therapies for certain clinical settings requiring short term blood replacement such as emergency resuscitation and hemodilution in surgery. Many advances have been made in developing these oxygen therapeutics, however safety concerns continue to slow their clinical progress. An important and often overlooked consideration in evaluating the safety of modified hemoglobins is the impact of chemical and/or genetic modifications on the redox chemistry of these proteins. Diaspirin cross-linked hemoglobin (DBBF-Hb) has been extensively evaluated in vitro and in animal models, and thus represents a useful model to explore possible correlations between structural-functional alterations and toxicity of hemoglobin-based blood substitutes.

Effects of hypoxia and glutathione depletion on hemoglobin- and myoglobin-mediated oxidative stress toward endothelium.

We investigated the toxicity of hemoglobin/myoglobin on endothelial cells under oxidative stress conditions that include cellular hypoxia and reduced antioxidant capacity. Bovine aorta endothelial cells (BAECs), grown on microcarrier beads, were subjected to cycles of hypoxia and reoxygenation in a small volume of medium, and endothelial cell monolayers were depleted of their intracellular glutathione (GSH) by treatment with buthionine sulfoximine. Incubation of diaspirin cross-linked hemoglobin (DBBF-Hb) or horse skeletal myoglobin (Mb) with BAECs subjected to 3 h of hypoxia caused transient oxidation of the hemoproteins to the ferryl form (Fe(4+)). Formation of the ferryl intermediate was decreased in a concentration-dependent manner by the addition of L-arginine, a substrate of NO synthase, after 3 h of hypoxia. Optimal inhibition of ferryl formation, possibly due to the antioxidant action of NO, was achieved with 900 microM L-arginine. Addition of hydrogen peroxide to GSH-depleted cells in the presence of DBBF-Hb or Mb significantly decreased cell viability. Ferryl Mb, but not ferryl DBBF-Hb, was observed in samples analyzed at the end of treatment, which may explain the greater toxicity observed with Mb as opposed to DBBF-Hb. This model may be utilized to identify causative agent(s) associated with hemoprotein cytotoxicity and in designing strategies to suppress or control heme-mediated injury under physiologically relevant conditions.

Detection of a ferrylhemoglobin intermediate in an endothelial cell model after hypoxia-reoxygenation.

A cell culture model of bovine aortic endothelial cells attached to microcarrier beads was used to study the interaction of diaspirin cross-linked hemoglobin (an oxygen-carrying blood substitute) with hypoxia-reoxygenation. Hemoglobin (200 microM) and hypoxia-volume restriction (3-5 h), together and separately, caused toxicity in this model, as measured by decreased cellular replating efficiency. Hemoglobin (60 microM) caused a reduction in hydrogen peroxide concentration and an increase in lipid peroxidation above that induced by hypoxia alone. Incubation of hemoglobin with endothelial cells caused transient oxidation of hemoglobin to its highly reactive and toxic ferryl species after >/=3 h of hypoxia, followed by 1 h of reoxygenation. Lipid peroxidation, which may occur in the presence of ferrylhemoglobin, also occurred after 1 h of reoxygenation. Hemoglobin caused a dose-dependent decrease in intracellular glutathione concentration, suggesting that it caused an oxidative stress to the cells. However, addition of ascorbate, alpha-tocopherol, or trolox did not decrease hemoglobin oxidation in the presence of normal or hypoxic cells. It is concluded that diaspirin cross-linked hemoglobin forms a ferryl intermediate in the absence of any exogenously added oxidant and contributes to the oxidative burden experienced by endothelial cells after hypoxia-reoxygenation, a condition that is likely to be encountered during trauma and surgery when hemoglobin solutions are used as perfusion agents.

Hemoglobin-based blood substitutes: oxygen carriers, pressor agents, or oxidants?

Hemoglobin-based blood substitutes are being developed as oxygen-carrying agents for the prevention of ischemic tissue damage and hypovolemic (low blood volume) shock. The ability of cell-free hemoglobin blood substitutes to affect vascular tone through the removal of nitric oxide has also prompted an evaluation of their usefulness for maintaining blood pressure in critically ill patients. Before the clinical potential of these substitutes can be fully realized, however, concerns remain as to the intrinsic toxicity of the hemoglobin molecule, particularly the interference of the heme prosthetic group with the tissue oxidant/antioxidant balance. This review provides some insights into the complex redox chemistry of hemoglobin and places an emphasis on how current knowledge may be exploited both to selectively enhance/suppress specific chemical reaction pathway(s) and to ultimately design safer hemoglobin-based therapeutics.

Cardiovascular and hemorheological effects of three modified human hemoglobin solutions in hemodiluted rabbits.

The cardiovascular effects of human albumin (Alb) and three human hemoglobin (Hb) solutions, dextran-benzene-tetracarboxylate Hb, alphaalpha-crosslinked Hb, and o-raffinose-polymerized Hb were compared in anesthetized rabbits undergoing acute isovolemic hemodilution with Hct reduction from 41.4 +/- 2.7 to 28.8 +/- 1.6%. The impact of the vasoconstricting properties of Hb was examined by measuring heart rate (HR), mean arterial pressure (MAP), abdominal aortic, and femoral arterial blood flow, vascular resistance (VR), and aortic distension during the first 3 h after hemodilution. The impact of the hemorheological parameters was assessed by measurements of hemodiluted blood viscosity. In contrast to Alb, the Hb solutions elicited an immediate increase in MAP (20-38%). The effects of Alb and Hb solutions on HR, as well as on aortic and femoral arterial blood flow, were similar. VR decreased with Alb (20-28%) and increased with all three Hb solutions (30-90%), but the MAP and VR rising trends were different with each Hb solution. Aortic distension decreased in Hb groups compared with the Alb group for the first 60 min. The viscosity of hemodiluted blood was similar for all groups at high shear rates but was dependent on the viscosity of the solutions at low shear rates. We conclude that the vasoconstriction elicited by the Hb solutions overrides the vasodilation associated with viscosity changes due to hemodilution and would be the major factor responsible to the cardiovascular changes.

Reactions of sperm whale myoglobin with hydrogen peroxide. Effects of distal pocket mutations on the formation and stability of the ferryl intermediate.

Distal pocket mutants of sperm whale oxymyoglobin (oxy-Mb) were reacted with a 2.5-fold excess of hydrogen peroxide (HOOH) in phosphate buffer at pH 7.0, 37 degreesC. We describe a mechanism composed of three distinct steps: 1) initial oxidation of oxy- to ferryl-Mb, 2) autoreduction of the ferryl intermediate to ferric metmyoglobin (metMb), and 3) reaction of metMb with an additional HOOH molecule to regenerate the ferryl intermediate creating a pseudoperoxidase catalytic cycle. Mutation of Leu-29(B10) to Phe slows the initial oxidation reaction 3-fold but has little effect on the rate of ferryl reduction to ferric met-aquo-myoglobin. In contrast, the Val-68(E11) to Phe mutation causes a small, 60% increase in the initial oxidation reaction and a much larger 2. 5-fold increase in the rate of autoreduction. Double insertion of Phe at both the B10- and E11-positions (L29F/V68F) produces a mutant with oxidation characteristics of both single mutants, slow initial oxidation, and rapid autoreduction, but an extraordinarily high affinity for O2. Replacing His-64(E7) with Gln produces 3-4-fold increases in both processes. Combining the mutation H64Q with L29F results in a myoglobin with enhanced resistance to metMb formation in the absence of antioxidant enzymes (i.e. catalase and superoxide dismutase) due to its own high pseudoperoxidase activity, which rapidly removes any HOOH produced in the initial stages of autoxidation. This double substitution occurs naturally in the myoglobin of Asian elephants, and similar multiple replacements have been used to reduce selectively the rate of nitric oxide (NO)-induced oxidation of both recombinant MbO2 and HbO2 blood substitute prototypes without altering O2 affinity.