Hb Nitric Oxide Research Articles

Cardiovascular effects of polymerized hemoglobin in a dyslipidemia model

The need for alternatives to allogeneic red blood cells (RBCs) for transfusion medicine has been recognized for more than a century. Large molecular size polymerized human hemoglobin (Hb) (PolyhHb) is a Hb based oxygen (O2) carrier (HBOC) recently evaluated with promising results in efficient O2 delivery and minimal toxicity. Despite significant commercial development, late stage clinical results of HBOC solutions hampered development. To evaluate the safety of PolyhHb as an O2 therapeutic agent in a vulnerable population, the present study was performed in a guinea pigs subjected to a high fat and high sucrose diet (HDHS). This model mimics human dyslipidemia and potentially induces endothelial dysfunction (ED). The objective of this study was to evaluate the impact of large molecular size PolyhHb on cardiovascular function in guinea pigs with dyslipidemia. Animals weighing between 150–200 g were fed a HDHS for 12 weeks. The metabolic characterization of the model included glucose tolerance testing (GTT) and measurement of the lipid profile. After metabolic characterization, the carotid artery and jugular vein were catheterized and catheters were exteriorized dorsally. Then, PolyhHb (n = 5) was subjected to a 20% blood volume (BV) exchange‐transfusion (BV estimated as 7.5% of body weight) with PolyhHb at 10 g/dL. Sham (n = 5) was subjected to the same procedure, but no exchange transfusion was performed. Post exchange, the animals were allowed to recover for 24 hours, after which blood pressure (BP) and heart rate (HR) were recorded, and BP and HR variability (HRV) were evaluated by spectral analysis. Animals were subjected to a sequence of vasoactive drugs to evaluate their baroreflex (phenylephrine, sodium nitroprusside, 1 and 2 ug), endothelial function (acethocoline (ACH), 2 and 4 ug), and nitric oxide response (Nitro‐L‐arginine methyl ester (L‐NAME), 12 mg/kg). Animals presented glucose intolerance and dyslipidemia. PolyhHb induced an 18% increase in MAP 24 hours after exchange‐transfusion. PolyhHb showed a decreased response to L‐NAME, suggesting a deficiency in nitric oxide (NO) dependent regulation of vascular tone, a known consequence of acellular Hb NO scavenging. There were no differences in baroreflex between groups nor significant endothelial dysfunction for the PolyhHb group compared to the Sham group. An increase in HRV and in sympathetic nervous system (SNS) modulation of the heart (low frequency band, LF) was observed in the PolyhHb group compared to Sham. On the other hand, parasympathetic modulation of the heart (high frequency band, HF) was decreased for the PolyhHb group compared to Sham, culminating in a 2‐fold increase in sympathetic vagal balance. Moreover, BP variability as well as blood vessel SNS modulation was increased for PolyhHb compared to Sham. In conclusion, PolyhHb transfusion impaired the autonomic nervous system in guinea pigs with dyslipidemia. Our results suggest that dyslipidemic patients might be more vulnerable to PolyhHb transfusion side effects.Support or Funding InformationThis work was supported by the NIH Heart Lung and Blood Institute under Grants T32‐HL105373, R01‐HL126945, and the DOD DMRDP under Grant W81XWH‐18‐1‐0059

Absence of Nonclassical Monocytes in Hemolytic Patients: Free Hb and NO-Mediated Mechanism.

In a recent work, we have described the kinetics among the monocyte subsets in the peripheral blood of hemolytic patients including paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell disease (SCD). After engulfing Hb-activated platelets, classical monocytes (CD14+CD16−) significantly transformed into highly inflammatory (CD14+CD16hi) subsets in vitro. An estimated 40% of total circulating monocytes in PNH and 70% in SCD patients existed as CD14+CD16hi subsets. In this study, we show that the nonclassical (CD14dimCD16+) monocyte subsets are nearly absent in patients with PNH or SCD, compared to 10-12% cells in healthy individuals. In mechanism, we have described the unique role of both free Hb and nitric oxide (NO) in reducing number of nonclassical subsets more than classical monocytes. After engulfing Hb-activated platelets, the monocytes including nonclassical subsets acquired rapid cell death within 12 h in vitro. Further, the treatment to monocytes either with the secretome of Hb-activated platelets containing NO and free Hb or purified free Hb along with GSNO (a physiological NO donor) enhanced rapid cell death. Besides, our data from both PNH and SCD patients exhibited a direct correlation between intracellular NO and cell death marker 7AAD in monocytes from the peripheral blood. Our data together suggest that due to the immune surveillance nature, the nonclassical or patrolling monocytes are encountered frequently by Hb-activated platelets, free Hb, and NO in the circulation of hemolytic patients and are predisposed to die rapidly.

The Impact of Surgery and Stored Red Blood Cell Transfusions on Nitric Oxide Homeostasis.

Cell-free hemoglobin (Hb) forms in stored red blood cells (RBCs) as a result of hemolysis. Studies suggest that this cell-free Hb may decrease nitric oxide (NO) bioavailability, potentially leading to endothelial dysfunction, vascular injury, and multiorgan dysfunction after transfusion. We tested the hypothesis that moderate doses of stored RBC transfusions increase cell-free Hb and decrease NO availability in postoperative surgical patients. Twenty-six patients undergoing multilevel spine fusion surgery were studied. We compared those who received no stored RBCs (n = 9) with those who received moderate amounts (6.1 ± 3.0 units) of stored RBCs over 3 perioperative days (n = 17). Percent hemolysis (cell-free Hb), RBC-NO (heme-NO), and plasma nitrite and nitrate were measured in samples from the stored RBC bags and from patients' blood, before and after surgery. Posttransfusion hemolysis was increased approximately 3.5-fold over preoperative levels (P = 0.0002) in blood samples collected immediately after surgery but not on postoperative days 1 to 3. Decreases in both heme-NO (by approximately 50%) and plasma nitrite (by approximately 40%) occurred postoperatively, both in nontransfused patients (P = 0.036 and P = 0.026, respectively) and transfused patients (P = 0.0068 and P = 0.003, respectively) and returned to preoperative baseline levels by postoperative day 2 or 3. Postoperative plasma nitrite and nitrate were decreased significantly in both groups, and this change was slower to return to baseline in the transfused patients, suggesting that blood loss and hemodilution from crystalloid administration contribute to this finding. The decrease in NO metabolites occurred irrespective of stored RBC transfusions, suggesting this decrease may be related to blood loss during surgery and hemodilution rather than to scavenging of NO or inhibition of NO synthesis by stored RBC transfusions.

Red blood cell membrane-facilitated release of nitrite-derived nitric oxide bioactivity.

The reduction of nitrite by deoxyhemoglobin to nitric oxide (NO) has been proposed as a mechanism for the transfer of NO bioactivity from the red blood cell (RBC) to the vasculature. This transfer can increase vascular dilatation. The major challenge to this hypothesis is the very efficient scavenging of NO by hemoglobin, which prevents the release of NO from RBCs. Previous studies indicate that the reaction of nitrite with deoxyhemoglobin produces two metastable intermediates involving nitrite bound to deoxyhemoglobin and a hybrid intermediate [Hb(II)NO(+) ↔ Hb(III)NO] where the nitrite is reduced, but unavailable to react with hemoglobin. We have now shown how unique properties of these intermediates provide a pathway for the release of NO bioactivity from RBCs. The high membrane affinity of these intermediates (>100-fold greater than that of deoxyhemoglobin) places these intermediates on the membrane. Furthermore, membrane-induced conformational changes of the nitrite-reacted intermediates facilitate the release of NO from the hybrid intermediate and nitrite from the nitrite-bound intermediate. Increased membrane affinity, coupled with facilitated dissociation of NO and nitrite from the membrane-bound intermediates, provides the first realistic mechanism for the potential release of NO and nitrite from the RBC and their potential transfer to the vasculature.

Oxygen binding to partially nitrosylated hemoglobin

Reactions of nitric oxide (NO) with hemoglobin (Hb) are important elements in protection against nitrosative damage. NO in the vasculature is depleted by the oxidative reaction with oxy Hb or by binding to deoxy Hb to generate partially nitrosylated Hb (Hb–NO). Many aspects of the formation and persistence of Hb–NO are yet to be clarified. In this study, we used a combination of EPR and visible absorption spectroscopy to investigate the interactions of partially nitrosylated Hb with O2. Partially nitrosylated Hb samples had predominantly hexacoordinate NO–heme geometry and resisted oxidation when exposed to O2 in the absence of anionic allosteric effectors. Faster oxidation occurred in the presence of 2,3-diphosphoglycerate (DPG) or inositol hexaphosphate (IHP), where the NO–heme derivatives had higher levels of pentacoordinate heme geometry. The anion-dependence of the NO–heme geometry also affected O2 binding equilibria. O2-binding curves of partially nitrosylated Hb in the absence of anions were left-shifted at low saturations, indicating destabilization of the low O2 affinity T-state of the Hb by increasing percentages of NO–heme, much as occurs with increasing levels of CO–heme. Samples containing IHP showed small decreases in O2 affinity, indicating shifts toward the low-affinity T-state and formation of inert α-NO/β-met tetramers. Most remarkably, O2-equilibria in the presence of the physiological effector DPG were essentially unchanged by up to 30% NO–heme in the samples. As will be discussed, under physiological conditions the interactions of Hb with NO provide protection against nitrosative damage without impairing O2 transport by Hb's unoccupied heme sites. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

P48: Effects of nitrite on hemodynamic parameters under hypoxic conditions

Blood loss and resuscitation after trauma are associated with decreased hematocrit and hypoxia. Previously we showed that nitric oxide (NO) supplemented resuscitation (Sobhian et al., Am. J. Surg. (2011)), despite a transient decrease in mean arterial pressure, significantly enhanced perfusion in various organs with a strong trend to improve survival. Recently nitrite was identified as a potent NO-source under hypoxia. The aim of this study was to investigate the hemodynamic effects of nitrite under hypoxic conditions in a volume replacement model in rats. Anesthetized rats were bled (0.5 min/ml) and Ringer’s solution was infused simultaneously (1.5 ml/min) to reduce hematocrit from 41 ± 2% to 20 ± 2%. Animals were randomly assigned to normoxia (NOX, PO2 > 80 mm Hg) or hypoxia (HOX, PO2 approx. 55 mm Hg). Hypoxia was induced by inhalation of nitrogen/air containing 15% O2. After 10 min of hypoxia or equal normoxic period, animals received a bolus dose of nitrite (15 μmol/kg). At given time points blood samples were taken for blood gas analysis and determination of NO–hemoglobin (NO–Hb) complexes. Hemodynamic parameters were determined by pulse wave analysis. Peripheral perfusion in the skin was analysed by laser Doppler imaging. Hypoxia led to a significant decrease in arterial pO2 (132.1 ± 7.0 vs. 58.9 ± 5.1 mm Hg) and SO2 (95.4 ± 0.4 vs. 69.1 ± 4.9%). Nitrite-derived NO assessed as NO–Hb complexes by electron paramagnetic resonance spectroscopy was 3.5 times higher in HOX compared to NOX group nitrite injection led to an immediate drop in mean arterial pressure (MAP) in both groups. While this was transient in the NOX group, in the HOX group MAP stayed significantly reduced until end ob observation (P < 0.05). In the HOX group vessel elasticity was significantly increased and vascular resistance decreased. This was associated with enhanced peripheral skin perfusion (P < 0.01). Similar to NO-supplemented resuscitation, nitrite under hypoxic conditions decreased MAP but increased peripheral perfusion. Additional studies will determine the impact on short-and long-term survival. Supported by FWF grant P21121. Supported by FWF Grant P21121.

P40: Platelet activation in the presence of nitrite, nitric oxide and heme

Background NO stimulates intercellular cGMP production leading to a reduction in platelet activation. Nitrite is reduced to nitric oxide (NO) by deoxygenated hemoglobin and therefore acts as a potential source of biological NO. Nitrite in the presence of red blood cells (RBCs) has been shown to reduce platelet aggregation and this reduction in aggregation is enhanced when RBCs are deoxygenated. In addition nitrite in the presences of deoxygenated RBCs has been shown to reduce platelet expression of p-selectin when platelets are activated by ADP. Methods Platelet rich plasma was separated from whole blood by centrifugation at 120 g for 10 min. Plasma was diluted 10-fold into phosphate buffered saline and exposed to nitrite, NO, and/or RBC, as indicated. Platelets were then activated by ADP After 10 min of incubation with ADP, platelets were labeled with fluorescent antibodies for platelet recognition (CD41 or CD61) and activation (Pac-1 or CD62P) and measured using flow cytometry. Results Here we confirm NO reduces platelet activation. However, in the presence of oxyhemoglobin (Hb) NO no longer reduces platelet activation. MetHb, which does not rapidly scavenge NO does not affect platelet activation reduction by NO. In our research we see similar trends in decreased platelet activation with increasing concentrations of nitrite in the presences on RBCs. However our results are not as sensitive to nitrite concentrations as previously published data. In addition we see trends of decreasing platelet activation with increasing nitrite concentration in the presence of Hb. Conclusions Our results verify the effect of NO on platelet activation. They also show a trend which agrees with previously published data showing a decrease in platelet activation in the presence of nitrite and red blood cells. Additionally we see the same trend in the presence of nitrite and cell free Hb. Disclosure Supported by NIH Grants HL058091 and HL098032.

Contribution of membrane permeability and unstirred layer diffusion to nitric oxide–red blood cell interaction

Nitric oxide (NO) consumption by red blood cell (RBC) hemoglobin (Hb) in vasculature is critical in regulating the vascular tone. The paradox of NO production at endothelium in close proximity of an effective NO scavenger Hb in RBCs is mitigated by lower NO consumption by RBCs compared to that of free Hb due to transport resistances including membrane resistance, extra- and intra-cellular resistances for NO biotransport to the RBC. Relative contribution of each transport resistance on NO–RBC interactions is still not clear. We developed a mathematical model of NO transport to a single RBC to quantify the contributions from individual transport barriers by analyzing the effect of RBC membrane permeability (Pm), hematocrit (Hct) and NO–Hb reaction rate constants on NO–RBC interactions. Our results indicated that intracellular diffusion of NO was not a rate limiting step for NO–RBC interactions. The extracellular diffusion contributed 70–90% of total transport resistance for Pm>1cms−1 whereas membrane resistance accounts for 50–75% of total transport resistance for Pm<0.1cms−1. We propose a narrow Pm range of 0.21–0.44cms−1 for 10–45% Hct, respectively, below which membrane resistance is more significant and above which extracellular diffusion is a dominating transport resistance for NO–RBC interactions.

Investigation of electrocatalytic pathway for hemoglobin toward nitric oxide by electrochemical approach based on protein controllable unfolding and in-situ reaction

An electrochemical approach based on protein controllable unfolding was developed and applied in combination with in-situ reaction in order to investigate the electrocatalytic pathway for hemoglobin (Hb) toward nitric oxide (NO). Hb was entrapped in a dimethyldidodecylammonium bromide (DDAB) film modified glassy carbon electrode (DDAB/Hb/GCE). Two typical denaturants of acid and urea were synergistically utilized to control the incorporated Hb to a most unfolded state without losing heme groups. Under optimal conditions, the unfolded DDAB/Hb/GCE exhibited accelerated direct electron transfer. The sensitivities for the detection of ascorbic acid (AA), NaNO2 and NO were improved as 3, 10 and 12 times higher than those on the native DDAB/Hb/GCE, and the limits of detection (LODs) for AA, NaNO2 and NO were down to 0.33, 0.83 and 0.063μM, respectively. The unfolded DDAB/Hb/GCE was further applied for the investigation of Hb–NO interaction in NaNO2 solution. With successive additions of AA, NO was generated in situ on DDAB/Hb/GCE. A new reduction peak of the intermediate HbFe(II)–HN2O2 was successfully revealed near −0.65V. The whole electrocatalytic mechanism was proposed and verified by density functional theory. The method can be a promising platform for facile study of the interaction between NO and heme proteins.

Nitrite increases NO-hemoglobin levels but does not affect vascular response under hypoxia

Nitric oxide (NO) released from nitrite under low oxygen tension may improve tissue perfusion and attenuate hypoxia induced injury due to its vasodilatory effect. We aimed to investigate the hemodynamic effects of nitrite under hypoxic and normoxic conditions in vivo. Anesthetized rats were bled and Ringer’s solution was infused simultaneously to reduce hematocrit to 20 ± 2%. Animals were randomly assigned to normoxia (NOX, pO2 >80 mmHg) or hypoxia (HOX, pO2 ∼55 mmHg). Hypoxia was induced by inhalation of nitrogen/air containing 15% O2. After 10 min of hypoxia or normoxia, animals received a bolus dose of nitrite (15 μmol/kg) resulting in an immediate drop in mean arterial blood pressure (MAP). Nitrite-derived NO assessed as NO–hemoglobin complex (NO–Hb) by electron paramagnetic resonance spectroscopy was 3.5 times higher in HOX compared to NOX group. Despite higher NO–Hb levels in HOX group, MAP response did not differ between groups (HOX 9.5 ± 1.9 vs. NOX 8.4 ± 3.3 mmHg). Similarly, vascular tone assessed by reflection index of pulse wave did not differ (HOX 71.5 ± 12.3 vs. NOX 82.1 ± 12.4). An increase in nitrite up to 50 μmol/kg resulted in further decrease in MAP suggesting that nitrite reduction had not reached a steady state level. Our results suggest that (i) nitrite mediated vasodilation is mediated by NO release from parenchymal rather than from red blood cells, (ii) oxygen tension in parenchymal cells under normoxic conditions is low enough to reduce nitrite to NO augmenting hemodynamic and vascular response similar to hypoxia. FWF grant P21121.

A New Paramagnetic Intermediate Formed During the Reduction of Nitrite by Deoxyhemoglobin

Nitric oxide (NO) plays a crucial role regulating vascular tone and blood flow. Recent studies have shown that RBCs can take up nitrite at reduced oxygen pressures to be reutilized by deoxyhemoglobin for the production of nitric oxide. However, it is still necessary to explain how NO bypasses hemoglobin scavenging. Our previous studies have suggested the reduction of nitrite back to NO by deoxyhemoglobin via intermediates that retain NO bioactivity. One of the intermediates characterized was a hybridized species with properties of Hb(II)NO+ and Hb(III)NO. The unexpected stability of this intermediate has been attributed to the transfer of an electron from the β-93 thiol of hemoglobin to NO+ producing •SHb(II)NO. The formation of this paramagnetic intermediate (•SHb(II)NO) was confirmed by studying the electron paramagnetic resonance (EPR) changes that take place during nitrite reduction by deoxyHb in comparison with deoxyHb treated with N-ethyl-maleimide (blocks the β-93 thiol group of hemoglobin) and carboxypeptidase A (removes C-terminus of hemoglobin), both of which stabilize the R-quaternary conformation of hemoglobin. The spectral changes observed were attributed to the equilibrium of the hybridized diamagnetic species with a paramagnetic species with an EPR spectrum that reflects that of NO bound to the heme in the β-chain plus that of a thiyl radical, •SHb(II)NO. Incubation studies at −20°C demonstrate that the intensity of this paramagnetic species decreases at this low temperature indicating a shift back to the hybrid intermediate, “Hb(II)NO+ ↔ Hb(III)NO”. This new paramagnetic intermediate species (•SHb(II)NO) explains the stability of the hybridized intermediate and the slow release of NO during the nitrite reduction. The involvement of a thiyl radical, at the same time provides a pathway for the formation of SNOHb that can also be a source for NO bioactivity.

Lipopolysaccharide (LPS) induction of nitric oxide synthase-2 and cyclooxygenase-2 is impaired in fructose overloaded rats

Aims Fructose (F) overload in rats induces metabolic dysfunctions that resemble the human metabolic syndrome. In this paper, we aimed to investigate the response of F overload rats to lipopolysaccharide (LPS) challenge in terms of nitric oxide (NO) production and prostanoids (PR) release. Main methods NO blood steady-state concentration was monitored through the detection of nitrosyl–hemoglobin complexes (NO–Hb) by electronic spin resonance. Production of 6-keto PGF 1α, PGE 2, PGF 2α and TXB 2 was measured in aorta and mesenteric beds by HPLC. Western blot analysis was used to examine the changes in the expression levels of NOS-2 and COX-2 in aorta. Key findings Our results showed that increases in NO circulating steady-state concentration and PR production by aorta and mesenteric beds 6 h after LPS administration were significantly attenuated in F overload rats with respect to control animals. Oxidative stress parameters were equally affected in the presence or absence of the F treatment. Aorta protein levels of NOS-2 and COX-2, two enzymes inducible by LPS, were significantly lower in F overload rats with respect to control rats at the end of the treatment (−39% and −61% for NOS-2 and COX-2 respectively). Significance These results suggest that the metabolic alterations established by 15 weeks of F overload should affect the response to LPS challenge due to an attenuation in the induction of NOS-2 and COX-2. This effect would be one of the components contributing to abnormalities in the course of the inflammatory response in other conditions associated to insulin resistance, such as diabetes.

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.

Quantification of Intermediates Formed during the Reduction of Nitrite by Deoxyhemoglobin

Nitric oxide (NO) plays a crucial role in human physiology by regulating vascular tone and blood flow. The short life-span of NO in blood requires a mechanism to retain NO bioactivity in the circulation. Recent studies have suggested a mechanism involving the reduction of nitrite back to NO by deoxyhemoglobin in RBCs. A role for RBCs in transporting NO must, however, bypass the scavenging of NO in RBCs by hemoglobin. To understand how the nitrite reaction can deliver bioactive NO to the vasculature, we have studied the intermediates formed during the reaction. A reliable measure of the total concentration of heme-associated nitrite/NO intermediates formed was provided by combining filtration to measure free nitrite by chemiluminescence and electron paramagnetic resonance to measure the final product Hb(II)NO. By modifying the chemiluminescence method used to detect NO, we have been able to identify two intermediates: 1) a heme-associated nitrite complex that is released as NO in acid solution in the presence of ascorbate and 2) an intermediate that releases NO at neutral pH in the presence of ferricyanide when reacted with an Fe(III) ligand like azide. This species designated as "Hb(II)NO(+) (<--)(-->)Hb(III)NO" has properties of both isomeric forms resulting in a slower NO dissociation rate and much higher stability than Hb(III)NO, but provides a potential source for bioactive NO, which can be released from the RBC. This detailed analysis of the nitrite reaction with deoxyHb provides important insights into the mechanism for nitrite induced vasodilation by RBCs.

Effect of Iron−Quercetin Complex on Reduction of Nitrite in in Vitro and in Vivo Systems

This study investigated whether reducing agents such as quercetin and iron(II) facilitate formation of nitric oxide (NO) gas from orally ingested nitrite in an vivo study. When 3 mg/kg Na (15)NO2 was orally administered to rats with or without iron(II) or quercetin, Hb (15)NO, which is indicative of systemic (15)NO, was detected in the blood, with the maximum blood concentration of Hb (15)NO at 15 min after nitrite or nitrite plus quercetin treatment, whereas after administration of nitrite plus iron(II) or nitrite plus iron(II) and quercetin, the time was shortened to 10 min. Interestingly, iron(II), quercetin, or iron(II) plus quercetin did not affect the total amount of Hb (15)NO generated from orally administered Na (15)NO2. However, the systemic nitrite concentration was significantly decreased in the presence of iron(II) or iron(II) plus quercetin. These results may indicate that iron(II) is critical to the generation of NO gas from nitrite, whereas quercetin contributed little under the in vivo experimental conditions.

Mixed haematopoietic chimerism for sickle cell disease prevents intravascular haemolysis

Mixed haematopoietic chimerism for sickle cell disease prevents intravascular haemolysis

Involvement of carbon monoxide produced by heme oxygenase in ABA-induced stomatal closure in Vicia faba and its proposed signal transduction pathway

Carbon monoxide (CO) has recently proven to be an important bioactive or signaling molecule in mammalian cells. Its effects are mainly mediated by nitric oxide (NO) and cyclic GMP (cGMP). In Vicia faba leaves, CO production and heme oxygenase (HO) activity, an important CO synthetic enzyme, are first reported to increase in response to ABA treatment, which could result in stomatal closure. Interestingly, ABA-induced stomatal closure in V. faba guard cells is partially blocked when the synthetic CO inhibitor ZnPP, or the CO/NO scavenger Hb is added. Furthermore, we show that, exogenously applied CO donor, hematin, and CO aqueous solution not only result in the enhancement of CO release, but also time-dependently induce stomatal closure, and the latter is mimicked by the application of an NO donor SNP. The above-mentioned stomatal closure effects are differentially reversed by the addition of tungstate, a potent inhibitor of NO synthetic enzyme nitrate reductase (NR), the specific NO scavenger cPTIO, ZnPP, or Hb. During treatment for 4 h, SNP, 0.01% CO aqueous solution or hematin significantly triggers NO synthesis, whereas cPTIO, or tungstate approximately fully inhibits NO fluorescence. Additionally, application of the GC inhibitor ODQ blocks CO-induced stomatal closure. This inhibition could be reversed when 8-Br-cGMP is added. Thus, the above results suggest that CO produced by HO is involved in ABA-induced stomatal closure, and NO and cGMP may function as downstream intermediates in the CO signaling responsible for stomatal closure.

The role of β93 Cys in the inhibition of Hb S fiber formation

Recent studies have suggested that nitric oxide (NO) binding to hemoglobin (Hb) may lead to the inhibition of sickle cell fiber formation and the dissolution of sickle cell fibers. NO can react with Hb in at least 3 ways: 1) formation of Hb(II)NO, 2) formation of methemoglobin, and 3) formation of S-nitrosohemoglobin, through nitrosylation of the β93 Cys residue. In this study, the role of β93 Cys in the mechanism of sickle cell fiber inhibition is investigated through chemical modification with N-ethylmaleimide. UV resonance Raman, FT-IR and electrospray ionization mass spectroscopic methods in conjunction with equilibrium solubility and kinetic studies are used to characterize the effect of β93 Cys modification on Hb S fiber formation. Both FT-IR spectroscopy and electrospray mass spectrometry results demonstrate that modification can occur at both the β93 and α104 Cys residues under relatively mild reaction conditions. Equilibrium solubility measurements reveal that singly-modified Hb at the β93 position leads to increased amounts of fiber formation relative to unmodified or doubly-modified Hb S. Kinetic studies confirm that modification of only the β93 residue leads to a faster onset of polymerization. UV resonance Raman results indicate that modification of the α104 residue in addition to the β93 residue significantly perturbs the α 1β 2 interface, while modification of only β93 does not. These results in conjunction with the equilibrium solubility and kinetic measurements are suggestive that modification of the α104 Cys residue and not the β93 Cys residue leads to T-state destabilization and inhibition of fiber formation. These findings have implications for understanding the mechanism of NO binding to Hb and NO inhibition of Hb S fiber formation.

Detection of Human Red Blood Cell-bound Nitric Oxide

Major disparities in reported levels of basal human nitric oxide metabolites have resulted in a recent literature focusing almost exclusively on methods. We chose to analyze triiodide chemiluminescence, drawn by the prospect of identifying why the most commonly employed assay in nitric oxide biology typically yielded lower metabolite values, compared with several other techniques. We found that the sensitivity of triiodide was greatly affected by the auto-capture of nitric oxide by deoxygenated cell-free heme in the reaction chamber. Potential contaminants and signal losses were also associated with standard sample purification procedures and the chemistry involved in nitrite removal. To inhibit heme nitric oxide auto-capture, we added potassium ferricyanide to the triiodide reagent, reasoning this would provide a more complete detection of any liberated nitric oxide. From human venous blood samples, we established nitric oxide levels ranging from 0.000178 to 0.00024 mol nitric oxide/mol hemoglobin. We went on to find significantly elevated nitric oxide levels in venous blood taken from diabetic patients in comparison to healthy controls (p < 0.0001). We concluded that the lack of signals reported of late by several groups using triiodide chemiluminescence for the detection of hemoglobin-bound nitric oxide may not represent levels on the border of assay sensitivity but rather underestimated values because of methodological limitations. We therefore stress the need for assay systems to be developed that differentiate between individual nitric oxide metabolite species and overcome the limitations we outline, allowing accurate conclusions to be drawn regarding physiological nitric oxide metabolite levels.

Erythrocyte membrane fluidity and haemoglobin haemoporphyrin conformation: features revealed in patients with heart failure

This study examined the possible involvement of abnormal erythrocyte oxygen (O 2) transport in the pathogenesis of heart failure. Haemoglobin (Hb) haemoporphyrin conformation was assessed by Raman spectroscopy (RS) of blood samples, whereas membrane fluidity was estimated at depths of 0.6–0.8 and 2.2 nm by electron-paramagnetic resonance spectroscopy of erythrocytes loaded with spin-labeled 5-doxylstearic acid and 16-doxylstearic acid, respectively. The fluidity of erythrocyte membranes from patients with heart failure was decreased in the area near the membrane surface and remained unchanged in the deeper hydrophobic membrane regions. The same differences were also detected in healthy controls subjected to chronic high-altitude hypoxia. RS demonstrated that in heart failure the total content of Hb–ligand complexes and the relative content of Hb–nitric oxide (NO) complexes with cleaved Fe 2+-globin bond was decreased, whereas content of Hb–NO complexes with preserved Fe 2+-globin bond was increased. We propose that this phenomenon contributes to the reduced O 2 tissue supply seen in patients with heart failure.