Efficacy of recombinant human Hb by 31P-NMR during isovolemic total exchange transfusion.

The ability of recombinant human Hb (rHb1.1), which is being developed as an oxygen therapeutic, to support metabolism was measured by in vivo 31P-NMR surface coil spectroscopy of the rat abdomen in control animals and in animals subjected to isovolemic exchange transfusion to hematocrit of <3% with human serum albumin or 5 g/dl rHb1.1. No significant changes in metabolite levels were observed in control animals for up to 6 h. The albumin-exchange experiments, however, resulted in a more than eightfold increase in Pi and a 50% drop in phosphocreatine and ATP within 40 min. The tissue pH dropped from 7.4 to 6.8. The decrease in high-energy phosphates obeyed Michaelis-Menten kinetics, with a Michaelis-Menten constant of 3% as the hematocrit at which a 50% drop in high-energy phosphates was observed. Exchange transfusion with rHb1.1 resulted in no significant drop in high-energy phosphates, no rise in Pi, and no change in tissue pH from 7.35 +/- 0.15 for up to 5 h after exchange. By these criteria, rHb1.1 at a plasma Hb concentration of approximately 5 g/dl after total exchange transfusion was able to sustain energy metabolism of gut tissue at levels indistinguishable from control rats with a threefold higher total Hb level in erythrocytes.     

Role of nitric oxide scavenging in vascular response to cell-free hemoglobin transfusion

Modified Hb solutions have been developed as O(2) carrier transfusion fluids, but of concern is the possibility that increased scavenging of nitric oxide (NO) within the plasma will alter vascular reactivity even if the Hb does not readily extravasate. The effect of decreasing hematocrit from approximately 30% to 18% by an exchange transfusion of a 6% sebacyl cross-linked tetrameric Hb solution on the diameter of pial arterioles possessing tight endothelial junctions was examined through a cranial window in anesthetized cats with and without a NO synthase (NOS) inhibitor. Superfusion of a NOS inhibitor decreased diameter, and subsequent Hb transfusion produced additional constriction that was not different from Hb transfusion alone but was different from the dilation observed by exchange transfusion of an albumin solution after NOS inhibition. In contrast, abluminal application of the cross-linked Hb produced constriction that was attenuated by the NOS inhibitor. Neither abluminal nor intraluminal cross-linked Hb interfered with pial arteriolar dilation to cromakalim, an activator of ATP-sensitive potassium channels. Pial vascular reactivity to hypocapnia and hypercapnia was unaffected by Hb transfusion. Microsphere-determined regional blood flow indicated selective decreases in perfusion after Hb transfusion in the kidney, small intestine, and neurohypophysis, which does not have tight endothelial junctions. Administration of a NOS inhibitor to reduce the basal level of NO available for scavenging before Hb transfusion prevented further decreases in blood flow to these regions compared with NOS inhibition alone. In contrast, blood flow to skeletal and left ventricular muscle increased, and cerebral blood flow was unchanged after Hb transfusion. This cross-linked Hb tetramer is known to appear in renal lymph but not in urine. We conclude that cell-free tetrameric Hb does not scavenge sufficient NO in the plasma space to significantly affect baseline tone in vascular beds with tight endothelial junctions but does produce substantial constriction in beds with porous endothelium. The data support increasing the molecular size of Hb by polymerization or conjugation to limit extravasation in all vascular beds to preserve normal vascular reactivity.     

Pericardial delivery of omega-3 fatty acid: a novel approach to reducing myocardial infarct sizes and arrhythmias

O2-carrying fluids based on hemoglobin (Hb) are in various stages of clinical trials to determine their suitability as O2-carrying plasma expanders. Polymerized Hb solutions are characterized by their vasoactivity, low O2 affinity, oncotic effect, prolonged shelf life, and stability. Physiological responses to facilitated O2 transport after exchange transfusion with polymerized bovine Hb (PBH) were studied in the hamster window chamber model during acute moderate anemia to determine how PBH affects microvascular perfusion and tissue oxygenation. The anemic state [29% hematocrit (Hct)] was induced by hemodilution with a plasma expander (70 kDa dextran). After hemodilution, animals were randomly assigned to different exchange transfusion groups. Study groups were based on the concentration of PBH used, namely: PBH at 13 g Hb/dl [PBH13], PBH diluted to 8 (PBH8) or 4 (PBH4) g Hb/dl in albumin solution at matching colloidal osmotic pressure (COP), and no PBH (only albumin solution) at matching COP (PBH0). Measurement of systemic parameters, microvascular hemodynamics, capillary perfusion, and intravascular and tissue O2 levels was performed at 18% Hct. Restitution of O2-carrying capacity with PBH13 increased arterial pressure and triggered vasoconstriction, low perfusion, and high peripheral resistance. PBH4 and PBH0 exhibited lower arterial pressures compared with PBH13. Exchange transfused animals with PBH8 and PBH4 better maintained perfusion and functional capillary density than PBH13. Blood gas parameters and acid-base balance were recovered proportional to microvascular perfusion. Arterial O2 tensions were improved with PBH4 and PBH8 by preventing O2 precapillary release and increasing O2 reserve. Further studies to establish PBH optimal dosage, efficacy, safety, and its effect on outcome are indicated before Hb-based O2-carrying blood substitutes are implemented in routine practice.     

Increased tissue PO2 and decreased O2 delivery and consumption after 80% exchange transfusion with polymerized hemoglobin

The O2-carrying blood substitute based on polymerized bovine hemoglobin (PBH) was used to determine efficacy in maintaining tissue Po2 after an 80% isovolemic blood exchange leading to a hematocrit of 19% [5.4 g Hb/dl from red blood cells (RBCs) and 6.3 g Hb/dl from PBH]. Effects were studied in terms of O2 delivery, O2 extraction, and tissue Po2 at the microcirculatory level at 1, 12, and 24 h after exchange transfusion in awake hamsters prepared with a window chamber model. At 1 h after exchange, arteriolar and venular diameters were decreased compared with baseline. Arteriolar diameter did not fully recover at 12 h after exchange, but venular diameter returned to normal. At 24 h after exchange, arteriolar and venular diameters were not different from baseline. Combining diameter and flow velocity data allowed us to calculate arteriolar and venular flows. At 1 h after exchange, arteriolar and venular flow was reduced compared with baseline. Arteriolar flow was lower at 12 h after exchange and recovered after 24 h. The number of capillaries with RBC passage [functional capillary density (FCD)] at 1 h after exchange with PBH was significantly lower than baseline. FCD remained decreased at 12 h; at 24 h after exchange transfusion, FCD was fully recovered. Tissue Po2 was maximal at 1 h after exchange and decreased progressively at 12 and 24 h after exchange. O2 release to the tissue was minimal at 1 h and increased at 12 and 24 h after exchange. These results suggest the impairment of tissue O2 metabolism after introduction of PBH into the circulation, which is mitigated as PBH concentration declines.     

Elimination of pyridoxylated polyhemoglobin after partial exchange transfusion in chimpanzees.

Partial exchange transfusion with 8.5% pyridoxylated polyhemoglobin solution [PolyHb-PPa] was performed in five male chimpanzees weighing 22-30 kg. Serial blood and urine samples were obtained for 3 days. Percutaneous liver biopsies were performed on the 3rd to 4th, and the 9th to 11th days after PolyHb-PPa administration. Mean exchange volume was 42.5 +/- 10.7 ml/kg BW (26.8-54.6 ml/kg), mean Hb dose 3.7 +/- 0.9 g PolyHb-PPa/kg BW (2.4-4.8 g/kg), mean exchange rate 56.7 +/- 7.1% (48.2-67.4%). All animals survived long-term. Analysis of the plasma Hb concentration-time data showed a first order decline at a plasma level of 3.7 +/- 0.9 g PolyHb-PPa/kg BW. Mean intravascular half-life was 14.6 +/- 3.2 h. Total renal elimination of PolyHb-PPa was about 7%. PolyHb-PPa was absorbed and stored by Kupffer cells and transformed into hemosiderin. Siderosis of Kupffer cells and renal tubules had largely subsided 10 days after PolyHb-PPa indicating subsequent in vivo degradation and metabolization of the polymerized Hb fractions.     

血浆置换及血小板输注治疗特发性血小板减少性紫癜疗效观察

摘要 目的：探究血浆置换及血小板输注治疗特发性血小板减少性紫癜疗效。方法：选择2016年2月至2019年1月于我院接受治疗的60例特发性血小板减少性紫癜患者为研究对象,按照其选择治疗方式的差异将其分为血小板输注组（20例）及血浆置换（Plasma exchange,PE）组（40例）,对比两组患者治疗有效率、治疗前后血细胞计数变化情况以及治疗中各类不良反应发生情况。结果：血小板输注组患者治疗显效数10例,有效数6例,总有效率80.00 %,PE组患者治疗显效数27例,有效数12例,治疗总有效率97.50 %,PE组治疗总有效率高于血小板输注组（P<0.05）。与治疗前比较,PE组患者的PLT、RBC计数和Hb水平出现了明显的升高,WBC计数出现明显的下降（P<0.05）,血小板输注组PLT、RBC计数和Hb水平也出现明显升高,WBC计数水平出现下降（P<0.05）,但组间比较显示治疗后PE组患者上述指标均优于血小板输注组（P<0.05）。血小板输注组患者不良反应总发生人数为4人,不良反应总发生率为20.00 %,PE组总不良反应发生人数3人,不良反应总发生率为7.50 %,PE组不良反应总发生率明显低于血小板输注组（P<0.05）。结论：血血浆置换及血小板输注治疗均对特发性血小板减少性紫癜具有较好的治疗效果,能够显著改善患者血细胞计数异常情况,但血浆置换治疗安全性更高。     

Blood volume and cardiac index in rats after exchange transfusion with hemoglobin-based oxygen carriers

Migita, Russell, Armando Gonzales, Maria L. Gonzales, Kim D. Vandegriff, and Robert M. Winslow. Blood volume and cardiac indexin rats after exchange transfusion with hemoglobin-based oxygencarriers. J. Appl. Physiol. 82(6):1995-2002, 1997.We have measured plasma volume and cardiac indexin rats after 50% isovolemic exchange transfusion with humanhemoglobin cross-linked between the -chains withbis(3,5-dibromosalicyl)fumarate (Hb) and with bovine hemoglobinmodified with polyethylene glycol (PEGHb). Hb and PEGHb differ incolloid osmotic pressure (23.4 and 118.0 Torr, respectively), oxygenaffinity (oxygen half-saturation pressure of hemoglobin = 30.0 and 10.2 Torr, respectively), viscosity (1.00 and 3.39 cP, respectively), andmolecular weight (64,400 and 105,000, respectively). Plasma volume wasmeasured by Evans blue dye dilution modified for interference by plasmahemoglobin. Blood volumes in PEGHb-treated animals were significantlyelevated (74.0 ± 3.5 ml/kg) compared with animals treated withHb (49.0 ± 1.2 ml/kg) or Ringer lactate (48.0 ± 2.0 ml/kg) or with controls (58.2 ± 1.9 ml/kg). Heart rate reductionafter Hb exchange is opposite to that expected with blood volumecontraction, suggesting that Hb may have a direct myocardialdepressant action. The apparently slow elimination of PEGHb during the2 h after its injection is a consequence of plasma volume expansion:when absolute hemoglobin (concentration × plasma volume) iscompared for PEGHb and Hb, no difference in their eliminationrates is found. These studies emphasize the need to understand bloodvolume regulation when the effects of cell-free hemoglobin onhemodynamic measurements are evaluated.

     

Hemodynamic effects after partial exchange transfusion with pyridoxylated polyhemoglobin in chimpanzees.

Partial exchange transfusion with 8.5% pyridoxylated polyhemoglobin solution [PolyHb-PPa] was performed in five anesthetized spontaneously-breathing male chimpanzees weighing 22-30 kg. Mean exchange volume was 42.5 +/- 10.7 ml/kg BW (26.8-54.6 ml/kg), mean exchange rate 56.7 +/- 7.1% (48.2-67.4%). All animals survived long-term. The chimpanzee's hemodynamics remained stable for the 5 h observation period. Right and left ventricular filling pressures remained constant, mean arterial pressure and mean pulmonary arterial pressure increased by up to 40% after the exchange. Cardiac output remained unaffected by the partial exchange and stroke volume did not change substantially although oxygen capacity and oxygen transport capacity decreased by about a third. The failure of cardiac output to rise after partial exchange transfusion with PolyHb-PPa contrasts with results after isovolemic hemodilution using non-oxygen-carrying blood substitutes and is not adequately explained by the oxygen capacity of 8.5% PolyHb-PPa (9.3 ml O2/dl).     

Oxygen transport by low and normal oxygen affinity hemoglobin vesicles in extreme hemodilution

The oxygen transport capacity of phospholipid vesicles encapsulating purified Hb (HbV) produced with a Po(2) at which Hb is 50% saturated (P 50 ) of 8 (HbV(8)) and 29 mmHg (HbV(29)) was investigated in the hamster chamber window model by using microvascular measurements to determine oxygen delivery during extreme hemodilution. Two isovolemic hemodilution steps were performed with 5% recombinant albumin (rHSA) until Hct was 35% of baseline. Isovolemic exchange was continued using HbV suspended in rHSA solution to a total [Hb] of 5.7 g/dl in blood. P(50) was modified by coencapsulating pyridoxal 5'-phosphate. Final Hct was 11% for the HbV groups, with a plasma [Hb] of 2.1 +/- 0.1 g/dl after exchange with HbV(8) or HbV(29). A reference group was hemodiluted to Hct 11% with only rHSA. All groups showed stable blood pressure and heart rate. Arterial oxygen tensions were significantly higher than baseline for the HbV groups and the rHSA group and significantly lower for the HbV groups compared with the rHSA group. Blood pressure was significantly higher for the HbV(8) group compared with the HbV(29) group. Arteriolar and venular blood flows were significantly higher than baseline for the HbV groups. Microvascular oxygen delivery and extraction were similar for the HbV groups but lower for the rHSA group (P < 0.05). Venular and tissue Po(2) were statistically higher for the HbV(8) vs. the HbV(29) and rHSA groups (P < 0.05). Improved tissue Po(2) is obtained when red blood cells deliver oxygen in combination with a high- rather than low-affinity oxygen carrier.     

Improved oxygenation in ischemic hamster flap tissue is correlated with increasing hemodilution with Hb vesicles and their O2 affinity

The aim of this study was to test the influence of oxygen affinity of Hb vesicles (HbVs) and level of blood exchange on the oxygenation in collateralized, ischemic, and hypoxic hamster flap tissue during normovolemic hemodilution. Microhemodynamics were investigated with intravital microscopy. Tissue Po2 was measured with Clark-type microprobes. HbVs with a P50 of 15 mmHg (HbV15) and 30 mmHg (HbV30) were suspended in 6% Dextran 70 (Dx70). The Hb concentration of the solutions was 7.5 g/dl. A stepwise replacement of 15%, 30%, and 50% of total blood volume was performed, which resulted in a gradual decrease in total Hb concentration. In the ischemic tissue, hemodilution led to an increase in microvascular blood flow to maximally 141-166% of baseline in all groups (median; P < 0.01 vs. baseline, not significant between groups). Oxygen tension was transiently raised to 121 +/- 17% after the 30% blood exchange with Dx70 (P < 0.05), whereas it was increased after each step of hemodilution with HbV15-Dx70 and HbV30-Dx70, reaching 217 +/- 67% (P < 0.01) and 164 +/- 33% (P < 0.01 vs. baseline and other groups), respectively, after the 50% blood exchange. We conclude that despite a decrease in total Hb concentration, the oxygenation in the ischemic, hypoxic tissue could be improved with increasing blood exchange with HbV solutions. Furthermore, better oxygenation was obtained with the left-shifted HbVs.     

A recombinant polymeric hemoglobin with conformational,functional, and physiological characteristics of an in vivo O2 transporter

With the objective of developing a recombinant oxygen carrier suitable for therapeutic applications, we have employed an Escherichia coli expression system to synthesize in high-yield hemoglobin (Hb) Minotaur, containing alpha-human and beta-bovine chains. Polymerization of Hb Minotaur through S-S intermolecular cross-linking was obtained by introducing a Cys at position beta9 and substituting the naturally occurring Cys. This homogeneous polymer, Hb Polytaur, has a molecular mass of approximately 500 kDa and was resistant toward reducing agents present in blood. In mice, the circulating half-time (3 h) was fivefold greater than adult human Hb (HbA). The half-time of autooxidation measured in blood (46 h) exceeded the circulating retention time. Hypervolemic exchange transfusion resulted in increased arterial blood pressure similar to that with albumin. The increase in pressure was less than that obtained by transfusion of cross-linked tetrameric Hb known to undergo renovascular extravasation. The nitric oxide reactivity of Hb Polytaur was similar to HbA, suggesting that the diminished pressor response to Hb Polytaur was probably related to diminished extravasation. Transfusion of 3% Hb Polytaur during focal cerebral ischemia reduced infarct volume by 22%. Therefore, site-specific Cys insertion on the Hb surface results in uniform size polymers that do not produce the large pressor response seen with tetrameric Hb. Polymerization maintains physiologically relevant oxygen and heme affinity, stability toward denaturation and oxidation, and effective oxygen delivery as indicated by reduced cerebral ischemic damage.     

Microvascular pressure and functional capillary density in extreme hemodilution with low- and high-viscosity dextran and a low-viscosity Hb-based O2 carrier

Blood losses are usually corrected initially by the restitution of volume with plasma expanders and subsequently by the restoration of oxygen-carrying capacity using either a blood transfusion or possibly, in the near future, oxygen-carrying plasma expanders. The present study was carried out to test the hypothesis that high-plasma viscosity hemodilution maintains perfused functional capillary density (FCD) by preserving capillary pressure. Microvascular pressure responses to extreme hemodilution with low- (LV) and high-viscosity (HV) plasma expanders and an exchange transfusion with a polymerized bovine cell-free Hb (PBH) solution were analyzed in the awake hamster window chamber model (n = 26). Systemic hematocrit was reduced from 50% to 11%. PBH produced a greater mean arterial blood pressure than the nonoxygen carriers. FCD was higher after a HV plasma expander (70 +/- 15%) vs. PBH (47 +/- 12%). Microvascular pressure spanning the capillary network was higher after a HV plasma expander (16-19 mmHg) compared with PBH (12-16 mmHg) and a LV plasma expander (11-14 mmHg) but lower than control (22-26 mmHg). FCD was found to be directly proportional to capillary pressure. The use of a HV plasma expander in extreme hemodilution maintained the number of perfused capillaries and tissue perfusion by comparison with a LV plasma expander due to increased mean arterial blood pressure and capillary pressure. The use of PBH increased mean arterial pressure but reduced capillary pressure due to vasoconstriction and did not maintain FCD.     

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.     

Effects of extreme hemodilution with hemoglobin-based O2 carriers on microvascular pressure

A surface-modified polyethylene glycol-conjugated human hemoglobin (MP4) and alpha alpha-cross-linked human hemoglobin (alpha alpha Hb) were used to restore oxygen carrying capacity in conditions of extreme hemodilution (hematocrit 11%) in the hamster window model preparation. Changes in microvascular function were analyzed in terms of effects on capillary pressure and functional capillary density (FCD). MP4, at 1.0 +/- 0.2 g/dl blood concentration, significantly lowered mean arterial pressure (MAP) below baseline (99.6 +/- 7.6 mmHg) to 82.4 +/- 6.9 mmHg (P < 0.05) and decreased of FCD to 70 +/- 9%. alpha alpha Hb caused a greater recovery in MAP to 94.4 +/- 6.2 mmHg and lowered FCD to 62 +/- 8%. However, differences between alpha alpha Hb and MP4 in FCD were not statistically significant. Capillary pressures were in the ranges of 17-21 mmHg for MP4 and 15-19 mmHg for alpha alpha Hb, with both significantly lower than baseline (P < 0.05). Pressure in 80-microm-diameter arterioles was significantly increased with alpha alpha Hb relative to MP4 (P < 0.05). These results were compared with previous findings on the relation between capillary pressure and FCD; they supported the concept of a relationship between FCD and capillary pressure. Measurement of changes in arteriolar diameter, microvascular blood flow, and FCD show that there was no statistical difference between using alpha alpha Hb and MP4 in extreme hemodilution. Microvascular resistance in arterioles with a diameter range of 70-80 microm showed an increase relative to control with alpha alpha Hb, whereas MP4 caused a decrease.     

Evidence for significant quantities of creatine kinase MM isoenzyme in human brain

Oxygen equilibrium studies have been carried out on hemoglobins A2 (alpha2delta2), Lepore-Washington (alpha2(deltabeta)2) and P-Nilotic (alpha2(beta2delta)2) using the beta chain containing hemoglobins A and S as controls. This investigation was initiated mainly because of controversial data that have been published on the oxygen affinity of hemoglobin (Hb) A2 and because samples containing the rare Hb P-Nilotic became available. Each hemoglobin was isolated in pure form by anion exchange chromatography; the samples used in the equilibrium analyses contained 100 mg Hb/dl with less than 5% ferrihemoglobin and no 2,3--diphosphoglycerate. Oxygen equilibrium analyses were made at 37 degrees C with the method of Benesch et al. (1965) Anal. Biochem. 11, 81--87; Anal. Biochem. 55, 245--248 (1973). A slight, but definite increase in oxygen affinity was observed for Hb A2 as well as for Hb P-Nilotic while the increase for the Hb Lepore-Washington was somewhat greater. The values for n, the Hill coefficient, and the Bohr effects were the same for all hemoglobin types. The differences in oxygen affinity of these hemoglobins apparently result from the differences in primary structure that are characteristic for those proteins.     

Peroxidase activity of hemoglobin towards ascorbate and urate: a synergistic protective strategy against toxicity of Hemoglobin-Based Oxygen Carriers (HBOC)

Acellular hemoglobins developed as oxygen bridging agents with volume expanding properties ("blood substitutes") are prone to autoxidation and oxidant-mediated structural changes in circulation. In the presence of hydrogen peroxide and either ascorbate or urate we show that ferric hemoglobin functions as a true enzymatic peroxidase. The activity saturates with both substrates and is linearly dependent on protein concentration. The activity is enhanced at low pH with a pKa of 4.7, consistent with protonation of the ferryl species (Fe(IV)-OH) as the active intermediate. To test whether these redox reactions define its behaviour in vivo we exchanged transfused guinea pigs with 50% polymerized bovine Hb (PolyHbBv) and monitored plasma levels of endogenous ascorbate and urate. Immediately after transfusion, met PolyHbBv levels increased up to 30% of total Hb and remained at this level during the first 24 h post transfusion. Plasma ascorbate decreased by 50% whereas urate levels remained unchanged after transfusion. A simple kinetic model, assuming that ascorbate was a more active ferric heme reductase and peroxidase substrate than urate, was consistent with the in vivo data. The present finding confirms the primary and secondary roles of ascorbate and urate respectively in maintaining the oxidative stability of infused Hb.     

Effects of amino acid substitutions at beta 131 on the structure and properties of hemoglobin: evidence for communication between alpha 1 beta 1- and alpha 1 beta 2-subunit interfaces

Substitutions of Asn, Glu, and Leu for Gln at the beta131 position of the hemoglobin molecule result in recombinant hemoglobins (rHbs) with moderately lowered oxygen affinity and high cooperativity compared to human normal adult hemoglobin (Hb A). The mutation site affects the hydrogen bonds present at the alpha(1)beta(1)-subunit interface between alpha103His and beta131Gln as well as that between alpha122His and beta35Tyr. NMR spectroscopy shows that the hydrogen bonds are indeed perturbed; in the case of rHb (beta131Gln --> Asn) and rHb (beta131Gln --> Leu), the perturbations are propagated to the other alpha(1)beta(1)-interface H-bond involving alpha122His and beta35Tyr. Proton exchange measurements also detect faster exchange rates for both alpha(1)beta(1)-interface histidine side chains of the mutant rHbs in 0.1 M sodium phosphate buffer at pH 7.0 than for those of Hb A under the same conditions. In addition, the same measurements in 0.1 M Tris buffer at pH 7.0 show a much slower exchange rate for mutant rHbs and Hb A. One of the mutants, rHb (beta131Gln --> Asn), shows the conformational exchange of its interface histidines, and exchange rate measurements have been attempted. We have also conducted studies on the reactivity of the SH group of beta93Cys (a residue located in the region of the alpha(1)beta(2)-subunit interface) toward p-mercuribenzoate, and our results show that low-oxygen-affinity rHbs have a more reactive beta93Cys than Hb A in the CO form. Our results indicate that there is communication between the alpha(1)beta(1)- and alpha(1)beta(2)-subunit interfaces, and a possible communication pathway for the cooperative oxygenation of Hb A that allows the alpha(1)beta(1)-subunit interface to modulate the functional properties in conjunction with the alpha(1)beta(2) interface is proposed.     

Reversal of hemoglobin-induced vasoconstriction with sustained release of nitric oxide

Erythrocyte free hemoglobin (Hb) induces vasoconstriction due to nitric oxide (NO) scavenging, limiting the NO available for vascular smooth muscle. The central objective of this study was to restore NO bioavailability using long-lived circulating NO-releasing nanoparticles (NO-np) to reverse the vasoconstriction and hypertension induced by polymerized bovine Hb (PBH) NO scavenging. PBH (13 g/dl) was infused in a volume equal to 10% of the animal blood volume. Intravascular NO supplementation was provided with an infusion of NO-np (10 and 20 mg/kg body wt). This study was performed using the hamster window chamber model to concurrently access systemic and microvascular hemodynamics. Infusion of PBH increased blood pressure and induced vasoconstriction. Treatment with 10 and 20 mg/kg NO-np reduced the blood pressure and vasoconstriction induced by PBH. Moreover, the higher dose of NO-np decreased blood pressure and induced vasodilation compared with baseline, respectively. Treatment with NO-np to decrease PBH-induced vasoconstriction increased methemoglobin levels and plasma nitrite and nitrate. In conclusion, NO-np counteracted both systemic hypertension and decreased the vasoconstrictor effects of PBH infusion, improving systemic and microvascular function. Based on the observed physiological properties, NO-np has clear potential as a therapeutic agent to replenish NO in situations where NO production is impaired, insufficient, or consumed, thereby preventing vascular complications.     

O2 release from Hb vesicles evaluated using an artificial,narrow O2-permeable tube: comparison with RBCs and acellular Hbs

A phospholipid vesicle that encapsulates a concentrated hemoglobin (Hb) solution and pyridoxal 5'-phosphate as an allosteric effector [Hb vesicle (HbV) diameter, 250 nm] has been developed to provide an O2 carrying ability to plasma expanders. The O2 release from flowing HbVs was examined using an O2-permeable, fluorinated ethylenepropylene copolymer tube (inner diameter, 28 microm) exposed to a deoxygenated environment. Measurement of O2 release was performed using an apparatus that consisted of an inverted microscope and a scanning-grating spectrophotometer with a photon-count detector, and the rate of O2 release was determined based on the visible absorption spectrum in the Q band of Hb. HbVs and fresh human red blood cells (RBCs) were mixed in various volume ratios at a Hb concentration of 10 g/dl in isotonic saline that contained 5 g/dl albumin, and the suspension was perfused at the centerline flow velocity of 1 mm/s through the narrow tube. The mixtures of acellular Hb solution and RBCs were also tested. Because HbVs were homogeneously dispersed in the albumin solution, increasing the volume of the HbV suspension resulted in a thicker marginal RBC-free layer. Irrespective of the mixing ratio, the rate of O2 release from the HbV/RBC mixtures was similar to that of RBCs alone. On the other hand, the addition of 50 vol% of acellular Hb solution to RBCs significantly enhanced the rate of deoxygenation. This outstanding difference in the rate of O2 release between the HbV suspension and the acellular Hb solution should mainly be due to the difference in the particle size (250 vs. 7 nm) that affects their diffusion for the facilitated O2 transport.