Synthesis and physicochemical characterization of a series of hemoglobin-based oxygen carriers: objective comparison between cellular and acellular types

A series of hemoglobin (Hb)-based O(2) carriers, acellular and cellular types, were synthesized and their physicochemical characteristics were compared. The acellular type includes intramolecularly cross-linked Hb (XLHb), polyoxyethylene (POE)-conjugated pyridoxalated Hb (POE-PLP-Hb), hydroxyethylstarch-conjugated Hb (HES-XLHb), and glutaraldehyde-polymerized XLHb (Poly-XLHb). The cellular type is Hb-vesicles (HbV) of which the surface is modified with POE (POE-HbV). Their particle diameters are 7 +/- 2, 22 +/- 2, 47 +/- 17, 68 +/- 24, and 224 +/- 76 nm, respectively, thus all the materials penetrate across membrane filters with 0.4 microm pore size, though only the POE-HbV cannot penetrate across the filter with 0.2 microm pore size. These characteristics of permeability are important to consider an optimal particle size in microcirculation in vivo. POE-PLP-Hb ([Hb] = 5 g/dL) showed viscosity of 6.1 cP at 332 s(-1) and colloid osmotic pressure (COP) of 70.2 Torr, which are beyond the physiological conditions (human blood, viscosity = 3-4 cP, COP = ca. 25 Torr). XLHb and Poly-XLHb showed viscosities of 1.0 and 1.5 cp, respectively, which are significantly lower than that of blood. COP of POE-HbV is regulated to 20 Torr in 5% human serum albumin (HSA). HES-XLHb and POE-HbV/HSA showed comparable viscosity with human blood. Microscopic observation of human red blood cells (RBC) after mixing blood with POE-PLP-Hb or HES-XLHb disclosed aggregates of RBC, a kind of sludge, indicating a strong interaction with RBC, which is anticipated to modify peripheral blood flow in vivo. On the other hand, XLHb and POE-HbV showed no rouleaux or aggregates of RBC. The acellular Hbs (P(50) = 14-32 Torr) have their specific O(2) affinities determined by their structures, while that of the cellular POE-HbV is regulated by coencapsulating an appropriate amount of an allosteric effector (e.g., P(50) = 18, 32 Torr). These differences in physicochemical characteristics between the acellular and cellular types indicate the advantages of the cellular type from the physiological points of view.     

Mechanisms of slower nitric oxide uptake by red blood cells and other hemoglobin-containing vesicles

Nitric oxide (NO) acts as a smooth muscle relaxation factor and plays a crucial role in maintaining vascular homeostasis. NO is scavenged rapidly by hemoglobin (Hb). However, under normal physiological conditions, the encapsulation of Hb inside red blood cells (RBCs) significantly retards NO scavenging, permitting NO to reach the smooth muscle. The rate-limiting factors (diffusion of NO to the RBC surface, through the RBC membrane or inside of the RBC) responsible for this retardation have been the subject of much debate. Knowing the relative contribution of each of these factors is important for several reasons including optimization of the development of blood substitutes where Hb is contained within phospholipid vesicles. We have thus performed experiments of NO uptake by erythrocytes and microparticles derived from erythrocytes and conducted simulations of these data as well as that of others. We have included extracellular diffusion (that is, diffusion of the NO to the membrane) and membrane permeability, in addition to intracellular diffusion of NO, in our computational models. We find that all these mechanisms may modulate NO uptake by membrane-encapsulated Hb and that extracellular diffusion is the main rate-limiting factor for phospholipid vesicles and erythrocytes. In the case of red cell microparticles, we find a major role for membrane permeability. These results are consistent with prior studies indicating that extracellular diffusion of several gas ligands is also rate-limiting for erythrocytes, with some contribution of a low membrane permeability.     

Estimation of nitric oxide concentration in blood for different rates of generation. Evidence that intravascular nitric oxide levels are too low to exert physiological effects

Endothelium-derived nitric oxide (NO) is a potent vasodilator in the cardiovascular system. Several lines of experimental evidence suggest that NO or NO equivalents may also be generated in the blood. However, blood contains a large amount of hemoglobin (Hb) in red blood cells (RBCs). The RBC-encapsulated Hb can react very quickly with NO, which is only limited by the rate of NO diffusion into the RBCs. It is unclear what the possible NO concentration levels in blood are and how the NO diffusion coefficient (D) and the permeability (Pm) of RBC membrane to NO affect the level of NO concentration. In this study, a steady-state concentration experimental method combined with a spherical diffusion model are presented for determining D and Pm and examining the effect of NO generation rate (V0) and hematocrit (Hct) on NO concentration. It was determined that Pm is 4.5 +/- 1.5 cm/s and D is 3410 +/- 50 microm2/s at 37 degrees C. Simulations based on experimental parameters show that, when the rate of NO formation is as high as 100 nm/s, the maximal NO concentration in blood is below 0.012 nM at Pm = 4.5 cm/s and Hct = 45%. Thus, it is unlikely that NO is directly exported or generated from the RBC as an intravascular signaling molecule, because its concentration would be too low to exert a physiological role. Furthermore, our results suggest that, if RBCs export NO bioactivity, this would be through NO-derived species that can release or form NO rather than NO itself.     

Functional coupling of oxygen binding and vasoactivity in S-nitrosohemoglobin

S-Nitrosohemoglobin (SNO-Hb) is a vasodilator whose activity is allosterically modulated by oxygen ("thermodyamic linkage"). Blood vessel contractions are favored in the oxygenated structure, and vasorelaxant activity is "linked" to deoxygenation, as illustrated herein. We further show that transnitrosation reactions between SNO-Hb and ambient thiols transduce the NO-related bioactivity, whereas NO itself is inactive. One remaining problem is that the amounts of SNO-Hb present in vivo are so large as to be incompatible with life were all the S-nitrosothiols transformed into bioactive equivalents during each arterial-venous cycle. Experiments were therefore undertaken to address how SNO-Hb conserves its NO-related activity. Our studies show that 1) increased O(2) affinity of SNO-Hb (which otherwise retains allosteric responsivity) restricts the hypoxia-induced allosteric transition that exchanges NO groups with ambient thiols for vasorelaxation; 2) some NO groups released from Cys(beta93) upon transition to T structure are autocaptured by the hemes, even in the presence of glutathione; and 3) an O(2)-dependent equilibrium between SNO-Hb and iron nitrosylhemoglobin acts to conserve NO. Thus, by sequestering a significant fraction of NO liberated upon transition to T structure, Hb can conserve NO groups that would otherwise be released in an untimely or deleterious manner.     

The Capacity of Red Blood Cells to Reduce Nitrite Determines Nitric Oxide Generation under Hypoxic Conditions

Nitric oxide (NO) is a key regulator of vascular tone. Endothelial nitric oxide synthase (eNOS) is responsible for NO generation under normoxic conditions. Under hypoxia however, eNOS is inactive and red blood cells (RBC) provide an alternative NO generation pathway from nitrite to regulate hypoxic vasodilation. While nitrite reductase activity of hemoglobin is well acknowledged, little is known about generation of NO by intact RBC with physiological hemoglobin concentrations. We aimed to develop and apply a new approach to provide insights in the ability of RBC to convert nitrite into NO under hypoxic conditions. We established a novel experimental setup to evaluate nitrite uptake and the release of NO from RBC into the gas-phase under different conditions. NO measurements were similar to well-established clinical measurements of exhaled NO. Nitrite uptake was rapid, and after an initial lag phase NO release from RBC was constant in time under hypoxic conditions. The presence of oxygen greatly reduced NO release, whereas inhibition of eNOS and xanthine oxidoreductase (XOR) did not affect NO release. A decreased pH increased NO release under hypoxic conditions. Hypothermia lowered NO release, while hyperthermia increased NO release. Whereas fetal hemoglobin did not alter NO release compared to adult hemoglobin, sickle RBC showed an increased ability to release NO. Under all conditions nitrite uptake by RBC was similar. This study shows that nitrite uptake into RBC is rapid and release of NO into the gas-phase continues for prolonged periods of time under hypoxic conditions. Changes in the RBC environment such as pH, temperature or hemoglobin type, affect NO release.     

Adaptive responses during anemia and its correction in lambs.

There is limited information available on which to base decisions regarding red blood cell (RBC) transfusion treatment in anemic newborn infants. Using a conscious newborn lamb model of progressive anemia, we sought to identify accessible metabolic and cardiovascular measures of hypoxia that might provide guidance in the management of anemic infants. We hypothesized that severe phlebotomy-induced isovolemic anemia and its reversal after RBC transfusion result in a defined pattern of adaptive responses. Anemia was produced over 2 days by serial phlebotomy (with plasma replacement) to Hb levels of 30-40 g/l. During the ensuing 2 days, Hb was restored to pretransfusion baseline levels by repeated RBC transfusion. Area-under-the-curve methodology was utilized for defining the Hb level at which individual study variables demonstrated significant change. Significant reciprocal changes (P < 0.05) of equivalent magnitude were observed during the phlebotomy and transfusion phases for cardiac output, plasma erythropoietin (Epo) concentration, oxygen extraction ratio, oxygen delivery, venous oxygen saturation, and blood lactate concentration. No significant change was observed in resting oxygen consumption. Cardiac output and plasma Epo concentration increased at Hb levels <75 g/l, oxygen delivery and oxygen extraction ratio decreased at Hb levels <60 g/l, and venous oxygen saturation decreased and blood lactate concentration increased at Hb levels <55 g/l. We speculate that plasma Epo and blood lactate concentrations may be useful measures of clinically significant anemia in infants and may indicate when an infant might benefit from a RBC transfusion.     

Encapsulation of hemoglobin inside liposomes surface conjugated with poly(ethylene glycol) attenuates their reactions with gaseous ligands and regulates nitric oxide dependent vasodilation

Acellular hemoglobin (Hb)-based O2 carriers (HBOCs) are being investigated as red blood cell (RBC) substitutes for use in transfusion medicine. However, commercial acellular HBOCs elicit both vasoconstriction and systemic hypertension which hampers their clinical use. In this study, it is hypothesized that encapsulation of Hb inside the aqueous core of liposomes should regulate the rates of NO dioxygenation and O2 release, which should in turn regulate its vasoactivity. To test this hypothesis, poly(ethylene glycol) (PEG) conjugated liposome-encapsulated Hb (PEG-LEHs) dispersions were prepared using human and bovine Hb. In this study, the rate constants for O2 dissociation, CO association, and NO dioxygenation were measured for free Hb and PEG-LEH dispersions using stopped-flow UV-visible spectroscopy, while vasoactivity was assessed in rat aortic ring strips using both endogenous and exogenous sources of NO. It was observed that PEG-LEH dispersions had lower O2 release and NO dioxygenation rate constants compared with acellular Hbs. However, no difference was observed in the CO association rate constants between free Hb and PEG-LEH dispersions. Furthermore, it was observed that Hb encapsulation inside vesicles prevented Hb dependent inhibition of NO-mediated vasodilation. In addition, the magnitude of the vasoconstrictive effects of Hb and PEG-LEH dispersions correlated with their respective rates of NO dioxygenation and O2 release. Overall, this study emphasizes the pivotal role Hb encapsulation plays in regulating gaseous ligand binding/release kinetics and the vasoactivity of Hb.     

Significance of affinity and cooperativity in oxygen binding to hemoglobin of horse fetal and maternal blood

The physiological significance of the position and shape of the oxygen equilibrium curve (OEC) of horse hemoglobin (Hb) is considered from the viewpoint of oxygen (O2) transport efficiency and the effectiveness of the Bohr effect. In horse fetal and maternal bloods, their physiological O2 affinities are nearly optimized with respect to the effectiveness of the Bohr shift occurring at the O2 release site, when it is measured by the change in O2 saturation per unit change in P50. With relatively low cooperativity (n=2.69) of horse Hb under physiological conditions, the effectiveness of the Bohr shift for fetal blood at O2 uptake site and maternal blood at O2 release site is high. These facts imply that the position and the cooperativity of horse Hb OEC are optimized to receive maximal benefit from the double Bohr shift. Before exercise, the position of the OEC for adult mares is nearly optimized for the effectiveness of the Bohr shift occurring at the O2 release site, whereas, at maximal exercise, the position of the OEC tends to become advantageous for O2 transport efficiency.     

Nitrite reductase activity of hemoglobin as a systemic nitric oxide generator mechanism to detoxify plasma hemoglobin produced during hemolysis

Hemoglobin (Hb) potently inactivates the nitric oxide (NO) radical via a dioxygenation reaction forming nitrate (NO(3)(-)). This inactivation produces endothelial dysfunction during hemolytic conditions and may contribute to the vascular complications of Hb-based blood substitutes. Hb also functions as a nitrite (NO(2)(-)) reductase, converting nitrite into NO as it deoxygenates. We hypothesized that during intravascular hemolysis, nitrite infusions would limit the vasoconstrictive properties of plasma Hb. In a canine model of low- and high-intensity hypotonic intravascular hemolysis, we characterized hemodynamic responses to nitrite infusions. Hemolysis increased systemic and pulmonary arterial pressures and systemic vascular resistance. Hemolysis also inhibited NO-dependent pulmonary and systemic vasodilation by the NO donor sodium nitroprusside. Compared with nitroprusside, nitrite demonstrated unique effects by not only inhibiting hemolysis-associated vasoconstriction but also by potentiating vasodilation at plasma Hb concentrations of <25 muM. We also observed an interaction between plasma Hb levels and nitrite to augment nitroprusside-induced vasodilation of the pulmonary and systemic circulation. This nitrite reductase activity of Hb in vivo was recapitulated in vitro using a mitochondrial NO sensor system. Nitrite infusions may promote NO generation from Hb while maintaining oxygen delivery; this effect could be harnessed to treat hemolytic conditions and to detoxify Hb-based blood substitutes.     

Contribution of cooperativity and the Bohr effect to efficient oxygen transport by hemoglobins from five mammalian species

By using published experimental values of the standard oxygen (O2) equilibrium curve and the in vivo arterial and venous O2 pressure (PO2) of fetal and maternal blood in five mammalian species (human, cow, pig, sheep, and horse), we investigated the relationship between the efficiency of O2 delivery and the effectiveness of the Bohr shift, and discussed the significance of cooperativity for mammalian Hb. The O2 delivery of fetal blood was more efficient than that of maternal blood, and the effectiveness of the Bohr shift at both O2 loading and release sites of fetal blood was high. A linear relationship was observed between the efficiency of O2 delivery and the effectiveness of the Bohr shift at O2 loading sites of the five mammalian species. In both fetal and maternal blood, the theoretically obtained optimal P50 value for O2 delivery (optP50(OD)) was nearly equal to the optimal P50 value for the effectiveness of the Bohr shift at the O2 loading site (optP50(BS)(loading)). This phenomenon was favorable for fetal blood to uptake O2 from maternal blood with the aid of the Bohr shift and to deliver a large amount of O2 to the tissues. The optP50s for the effectiveness of the Bohr shift at given arterial PO2 (PaO2) and venous PO2 (PvO2) were derived as follows: optP50(BS)(loading) = PaO2((n+1)/(n-1))(1/n), and optP50(BS)(release) = PvO2((n+1)/(n-1))(1/n). The relationship between in vivo PO2s and n, PaO2/PvO2 = ((n+1)/(n-1))(2/n), was derived by letting optP50 for the efficiency of O2 delivery be equal to that for the effectiveness of the Bohr shift.     

Effects of the RBC membrane and increased perfusate viscosity on hypoxic pulmonary vasoconstriction.

Red blood cells (RBCs) augment hypoxic pulmonary vasoconstriction (HPV) in part by scavenging of nitric oxide (NO) by Hb (Deem S, Swenson ER, Alberts MK, Hedges RG, and Bishop MJ, Am J Respir Crit Care Med 157: 1181-1186, 1998). We studied the contribution of the RBC compartmentalization of Hb to augmentation of HPV and scavenging of NO in isolated perfused rabbit lungs. Lungs were initially perfused with buffer; HPV was provoked by a 5-min challenge with hypoxic gas (inspired O(2) fraction 0.05). Expired NO was measured continuously. Addition of free Hb to the perfusate (0.25 mg/ml) resulted in augmentation of HPV and a fall in expired NO that were similar in magnitude to those associated with a hematocrit of 30% (intracellular Hb of 100 mg/ml). Addition of dextran resulted in a blunting of HPV after free Hb but no change in expired NO. Blunting of HPV by dextran was not prevented by NO synthase inhibition with N(omega)-nitro-L-arginine and/or cyclooxygenase inhibition. RBC ghosts had a mild inhibitory effect on HPV but caused a small reduction in expired NO. In conclusion, the RBC membrane provides a barrier to NO scavenging and augmentation of HPV by Hb. Increased perfusate viscosity inhibits HPV by an undetermined mechanism.     

Diffusing capacity reexamined: relative roles of diffusion and chemical reaction in red cell uptake of O2, CO, CO2, and NO.

This paper presents an analytical expression for the diffusing capacity (Theta(t)) of the red blood cell (RBC) for any reactive gas in terms of size and shape of the RBC, thickness of the unstirred plasma layer surrounding the RBC, diffusivities and solubilities of the gas in RBC and boundary layer, hematocrit, and the slope of the dissociation curve. The expression for Theta(t) has been derived by spatial averaging of the fundamental convection-diffusion-reaction equation for O(2) in the RBC and has been generalized to all cell shapes and for other reactive gases such as CO, NO, and CO(2). The effects of size and shape of the RBC, thickness of the unstirred plasma layer, hemoglobin concentration, and hematocrit on Theta(t) have been analyzed, and the analytically obtained expression for Theta(t) has been validated by comparison with different sets of existing experimental data for O(2) and CO(2). Our results indicate that the discoidal shape of the human RBC with average dimensions of 1.6-mum thickness and 8-mum diameter is close to optimal design for O(2) uptake and that the true reaction velocity in the RBC is suppressed significantly by the mass transfer resistance in the surrounding unstirred layer. In vitro measurements using rapid-mixing technique, which measures Theta(t) in the presence of artificially created large boundary layers, substantially underpredicts the in vivo diffusing capacity of the RBC in the diffusion-controlled regime. Depending on the conditions in the RBC, uptake of less reactive gases (such as CO) undergoes transition from reaction-limited to diffusion-limited regime. For a constant set of morphological parameters, the theoretical expression for Theta(t) predicts that Theta(t,NO) > Theta(t,)(CO(2)) > Theta(t,)(O(2)) > Theta(t,CO).     

Blood rheology of Weddell seals and bowhead whales

Red blood cell (RBC) aggregation and blood viscosity are important determinants of in vivo blood flow dynamics and, in marine mammals, these parameters may impact diving physiology by altering blood oxygen delivery during the diving response. Weddell seals are superb divers and exhibit age-related patterns in blood oxygen chemistry and diving ability. By contrast, bowhead whales are not long duration divers, and little is known of their blood properties relative to diving. The present study was designed to compare rheological characteristics of blood from Weddell seal pups, Weddell seal adults, and from adult bowhead whales: blood viscosity and RBC aggregation in plasma and in polymer solutions (i.e., RBC "aggregability") were measured. Salient findings included: (1) significant 4- to 8-fold greater aggregation in blood from adult seals compared with pups and human subjects; (2) 2-to 8-fold greater aggregation in bowhead whale blood compared with human blood; (3) compared to human red cells, enhanced RBC aggregability of RBC from adult seals and whales as determined by their greater aggregation in polymer solutions; (4) increasing RBC aggregation and aggregability of seal pup blood over a seven day period following birth; (5) significantly greater blood viscosity for adult seals compared with pups at both native and standardized hematocrits. These results indicate that, for both species, hemorheological parameters differ markedly from those of humans, and suggest progressive changes with seal age; the physiological implications of these differences have yet to be fully defined.     

Effect of nitric oxide on the transport and release of oxygen in fetal blood

It is well known that nitric oxide (NO), the most important vasodilator agent, plays an important role in lowering vascular resistance in the human umbilical-placental circulation and that its deficiency is related to the pathogenesis of pre-eclamptic disorder. Besides it has recently been demonstrated that human hemoglobin (HbA) is able to transport nitric oxide, as S-nitrosohemoglobin (SNO-Hb), from the arterial to the venous blood. In the present study we examine the functional properties of the adult and fetal nitrosated hemoglobins to see if the double transport of oxygen and NO may influence the fetal oxygenation and the relation between maternal and fetal blood. Our results show that S-nitrosation significantly increases the oxygen affinity of the adult Hb (HbA) with respect to native protein (no-nitrosated) while the functional properties of HbF are less influenced. The oxygen affinity modification, found for SNO-HbA, was ascribed to the nitrosation of cysteine beta 93: really, the same residue is also present in the gamma chains of fetal hemoglobin, while the increase of affinity is less evidenced; hence, it is probable that the 39 aminoacidic substitutions between beta and gamma chains allay the effects of S-nitrosation. As regards the physiological modulators (protons, chloride ions, 2,3-diphosphoglyceric acid, and temperature), they influence the oxygen affinity of the two hemoglobins S-nitrosated, in equal mode with respect to the native forms determining the same variation on the oxygen affinity. Hence, our results evidence the fact that the NO release by SNO-HbA "in vivo" would be limited to regions of extremely low oxygen tension (such as hypoxic regions), while in fetus, SNO-HbF would unload nitric oxide and oxygen at pressure values close to normal.     

Nitric oxide uptake by erythrocytes is primarily limited by extracellular diffusion not membrane resistance

The process of NO transfer into erythrocytes (RBCs) is of critical biological importance because it regulates the bioavailability and diffusional distance of endothelial-derived NO. It has been reported that the rate of NO reaction with oxyhemoglobin (Hb) within RBCs is nearly three orders of magnitude slower than that by equal amounts of free oxyhemoglobin. Consistent with early studies on oxygen uptake by RBCs, the process of extracellular diffusion was reported to explain this much lower NO uptake by RBC encapsulated Hb (Liu, X., Miller, M. J., Joshi, M. S., Sadowska-Krowicka, H., Clark, D. A., and Lancaster, J. R., Jr. (1998) J. Biol. Chem. 273, 18709-18713). However, it was subsequently proposed that the RBC membrane provides the main resistance to NO uptake rather than the process of extracellular diffusion (Vaughn, M. W., Huang, K. T., Kuo, L., and Liao, J. C. (2000) J. Biol. Chem. 275, 2342-2348). This conclusion was based on competition experiments that were assumed to be able to determine the rate constant of NO uptake by RBCs without extracellular diffusion limitation. To test the validity of this hypothesis, we theoretically analyzed competition experiments. Here, we show that competition experiments do not eliminate the extracellular diffusion limitation. Simulation of the competition data indicates that the main resistance to NO uptake by RBCs is caused by extracellular diffusion in the unstirred layer surrounding each RBC but not by the RBC membrane. This extracellular diffusion resistance is responsible for preventing interference of NO signaling in the endothelium without the need for special NO uptake by intracellular hemoglobin or a unique membrane resistance mechanism.     

Oxygen binding and oxidation reactions of human hemoglobin conjugated to carboxylate dextran

Human hemoglobin (Hb) conjugated to benzene tetracarboxylate substituted dextran produces a polymeric Hb (Dex-BTC-Hb) with similar oxygen affinity to that of red blood cells (P(50)=28-29 mm Hg). Under physiological conditions, the oxygen affinity (P(50)) of Dex-BTC-Hb is 26 mm Hg, while that of native purified human HbA(0) is 14 mm Hg, but it exhibits a slight reduction in cooperativity (n(50)), Bohr effect, and lacks sensitivity to inositol hexaphosphate (IHP), when compared to HbA(0). Oxygen-binding kinetics, measured by rapid mixing stopped-flow method showed comparable oxygen dissociation and association rates for both HbA(0) and Dex-BTC-Hb. The rate constant for NO-mediated oxidation of the oxy form of Dex-BTC-Hb, which is governed by NO entry to the heme pocket, was reduced to half of the value obtained for HbA(0). Moreover, Dex-BTC-Hb is only slightly more sensitive to oxidative reactions than HbA(0), as shown by about 2-fold increase in autoxidation, and slightly higher H(2)O(2) reaction and heme degradation rates. Dextran-BTC-based modification of Hb produced an oxygen-carrying compound with increased oxygen release rates, decreased oxygen affinity and reduced nitric oxide scavenging, desirable properties for a viable blood substitute. However, the reduction in the allosteric function of this protein and the lack of apparent quaternary T-->R transition may hinder its physiological role as an oxygen transporter.     

Interaction of nitrogen monoxide with hemoglobin and the artefactual production of S-nitroso-hemoglobin

Hemoglobin (Hb) is probably the most thoroughly studied protein in the human body. However, it has recently been proposed that in addition to the well known function of dioxygen and carbon dioxide transporter, one of the main roles of hemoglobin is to store and transport nitrogen monoxide. This hypothesis is highly disputed and is in contrast to the proposal that hemoglobin serves as an NO. scavenger in the blood. In this short review, I have presented the current status of research on the much-debated mechanism of the reaction between circulating hemoglobin and NO.. Despite the fact that oxyHb is extremely rapidly oxidized by NO., under basal physiological conditions the biological activity of NO. in the blood vessels is not completely lost. It has been shown that three factors reduce the efficiency of hemoglobin to scavenge NO.: a so-called red blood cell-free zone created close to the vessel wall by intravascular flow, an undisturbed layer around the red blood cells--where the NO. concentration is much smaller than the bulk concentration--and/or the red blood cell membrane. Alternatively, it has been proposed that NO. binds to Cys beta 93 of oxyHb, is liberated after deoxygenation of Hb, and consequently allows for a more effective delivery of O2 to peripheral tissues. However, because of the extremely fast rate of the reaction between NO. and oxyHb, experiments in vitro lead to artefactual production of large amounts of S-nitroso-hemoglobin. These results, together with other data, which challenge most steps of the NO.-transporter hypothesis, are discussed.     

Mechanism of oxidative damage to fish red blood cells by ozone

The present study was conducted to elucidate the adverse effects of ozone exposure on rainbow trout (Oncorhynchus mykiss) red blood cells (RBCs). We evaluated whether hemoglobin (Hb) or Hb-derived free iron could participate in the RBC damage using an in vitro ozone exposure system. Ozone exposure induced hemolysis, formation of methemoglobin, and RBC membrane lipid peroxidation. This RBC damage was not suppressed by the addition of a specific iron chelator (deferoxamine mesilate) to the medium but was suppressed by carbon monoxide (CO) treatment before ozone exposure. Generation of hydrogen peroxide (H2O2) in RBC was observed upon ozone exposure but was significantly suppressed by CO treatment before ozone exposure. Thus the Hb status (i.e., Hb redox condition) and H2O2 generation in RBC should play important roles in mediating RBC damage by ozone exposure. In other words, neither ozone nor its derivative directly attacked from the outside of the cell, but ozone that penetrated through the membrane derived the reactive oxygen species from Hb inside of the cell.     

Antioxidants inhibit angiogenesis in vivo through down-regulation of nitric oxide synthase expression and activity

Although reactive oxygen species (ROS) participate in many cellular mechanisms, only few data exist concerning their involvement in physiological angiogenesis. The aim of the present work was to elucidate possible mechanisms through which ROS affect angiogenesis in vivo, using the model of the chicken embryo chorioallantoic membrane (CAM). Superoxide dismutase (SOD) and its membrane permeable mimetic tempol, dose dependently decreased angiogenesis and down-regulated inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. The NADPH oxidase inhibitors, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) and apocynin, but not allopurinol, also had a dose dependent inhibitory effect on angiogenesis and NO production in vivo. Catalase and the intracellular hydrogen peroxide (H2O2) scavenger sodium pyruvate decreased, while H2O2 increased in a dose-dependent manner the number of CAM blood vessels, as well as the expression and activity of iNOS. Dexamethasone, which down-regulated NO production by iNOS and L-NAME, but not D-NAME, dose dependently decreased angiogenesis in vivo. These data suggest that antioxidants affect physiological angiogenesis in vivo, through regulation of NOS expression and activity.     

Hemoglobin-vesicles for a transfusion alternative and targeted oxygen delivery

Hb-vesicles (HbV) are artificial oxygen carriers that encapsulate purified Hb solution (35 g/dl) in unilamellar phospholipid vesicles (liposomes). The dispersion stability of HbV is attained using surface-modification with polyethylene glycol (PEG), so that the deoxygenated HbV can be stored at room temperature for years. Moreover, the intravenously injected HbV does not induce aggregation when contacted with blood components. Animal experiments have verified the safety and efficacy of HbV as a transfusion alternative. One advantage of HbV is that the O(2) affinity (P(50)) of HbV can be regulated easily to that of RBC (28 torr) and to other values by manipulating the amount of the allosteric effectors, such as pyridoxal 5'-phosphate, coencapsulated in HbV. It is possible that HbV with a lower P(50) (higher O(2) affinity) would retain O(2) in the normal tissue while unloading O(2) to a targeted hypoxic tissue. Small HbV (250-280 nm diameter) is distributed homogeneously in the plasma phase, and HbV would transport oxygen through collateral arteries in the ischemic tissues. Results of in vitro and in vivo experiments of the domestic and international collaborations have confirmed the possibility of targeted O(2) delivery by HbV.