S-nitrosohemoglobin Research Articles

Renal dysfunction in adults following cardiopulmonary bypass is linked to declines in S-nitroso hemoglobin: a case series.

Impaired kidney function is frequently observed in patients following cardiopulmonary bypass (CPB). Our group has previously linked blood transfusion to acute declines in S-nitroso haemoglobin (SNO-Hb; the main regulator of tissue oxygen delivery), reductions in intraoperative renal blood flow, and postoperative kidney dysfunction. While not all CPB patients receive blood, kidney injury is still common. We hypothesized that the CPB procedure itself may negatively impact SNO-Hb levels leading to renal dysfunction. After obtaining written informed consent, blood samples were procured immediately before and after CPB, and on postoperative day (POD) 1. SNO-Hb levels, renal function (estimated glomerular filtration rate; eGFR), and plasma erythropoietin (EPO) concentrations were quantified. Additional outcome data were extracted from the patients' medical records. Twenty-seven patients were enroled, three withdrew consent, and one was excluded after developing bacteremia. SNO-Hb levels declined after surgery and were directly correlated with declines in eGFR (R=0.48). Conversely, plasma EPO concentrations were elevated and inversely correlated with SNO-Hb (R=-0.53) and eGFR (R=-0.55). Finally, ICU stay negatively correlated with SNO-Hb concentration (R=-0.32). SNO-Hb levels are reduced following CPB in the absence of allogenic blood transfusion and are predictive of decreased renal function and prolonged ICU stay. Thus, therapies directed at maintaining or increasing SNO-Hb levels may improve outcomes in adult patients undergoing cardiac surgery.

Control of tissue oxygenation by S-nitrosohemoglobin in human subjects

S-Nitrosohemoglobin (SNO-Hb) is unique among vasodilators in coupling blood flow to tissue oxygen requirements, thus fulfilling an essential function of the microcirculation. However, this essential physiology has not been tested clinically. Reactive hyperemia following limb ischemia/occlusion is a standard clinical test of microcirculatory function, which has been ascribed to endothelial nitric oxide (NO). However, endothelial NO does not control blood flow governing tissue oxygenation, presenting a major quandary. Here we show in mice and humans that reactive hyperemic responses (i.e., reoxygenation rates following brief ischemia/occlusion) are in fact dependent on SNO-Hb. First, mice deficient in SNO-Hb (i.e., carrying C93A mutant Hb refractory to S-nitrosylation) showed blunted muscle reoxygenation rates and persistent limb ischemia during reactive hyperemia testing. Second, in a diverse group of humans-including healthy subjects and patients with various microcirculatory disorders-strong correlations were found between limb reoxygenation rates following occlusion and both arterial SNO-Hb levels (n = 25; P= 0.042) and SNO-Hb/total HbNO ratios (n = 25; P= 0.009). Secondary analyses showed that patients with peripheral artery disease had significantly reduced SNO-Hb levels and blunted limb reoxygenation rates compared with healthy controls (n = 8 to 11/group; P< 0.05). Low SNO-Hb levels were also observed in sickle cell disease, where occlusive hyperemic testing was deemed contraindicated. Altogether, our findings provide both genetic and clinical support for the role of red blood cells in a standard test of microvascular function. Our results also suggest that SNO-Hb is a biomarker and mediator of blood flow governing tissue oxygenation. Thus, increases in SNO-Hb may improve tissue oxygenation in patients with microcirculatory disorders.

Optimized S-nitrosohemoglobin Synthesis in Red Blood Cells to Preserve Hypoxic Vasodilation Via βCys93.

Classic physiology links tissue hypoxia to oxygen delivery through control of microvascular blood flow (autoregulation of blood flow). Hemoglobin (Hb) serves both as the source of oxygen and the mediator of microvascular blood flow through its ability to release vasodilatory S-nitrosothiol (SNO) in proportion to degree of hypoxia. β-globin Cys93Ala (βCys93Ala) mutant mice deficient in S-nitrosohemoglobin (SNO-Hb) show profound deficits in microvascular blood flow and tissue oxygenation that recapitulate microcirculatory dysfunction in multiple clinical conditions. However, the means to replete SNO in mouse red blood cells (RBCs) to restore RBC function is not known. In particular, although methods have been developed to selectively S-nitrosylate βCys93 in human Hb and intact human RBCs, conditions have not been optimized for mouse RBCs that are used experimentally. Here we show that loading SNO onto Hb in mouse RBC lysates can be achieved with high stoichiometry and β-globin selectivity. However, S-nitrosylation of Hb within intact mouse RBCs is ineffective under conditions that work well with human RBCs, and levels of metHb are prohibitively high. We developed an optimized method that loads SNO in mouse RBCs to maintain vasodilation under hypoxia and shows that loss of SNO loading in βCys93Ala mutant RBCs results in reduced vasodilation. We also demonstrate that differences in SNO/met/nitrosyl Hb stoichiometry can account for differences in RBC function among studies. RBCs loaded with quasi-physiologic amounts of SNO-Hb will produce vasodilation proportionate to hypoxia, whereas RBCs loaded with higher amounts lose allosteric regulation, thus inducing vasodilation at both high and low oxygen level. SIGNIFICANCE STATEMENT: Red blood cells from mice exhibit poor hemoglobin S-nitrosylation under conditions used for human RBCs, frustrating tests of vasodilatory activity. Using an optimized S-nitrosylation protocol, mouse RBCs exhibit hypoxic vasodilation that is significantly reduced in hemoglobin βCys93Ala mutant RBCs that cannot carry S-nitrosothiol allosterically, providing genetic validation for the role of βCys93 in oxygen delivery.

Hypoxic vasodilatory defect and pulmonary hypertension in mice lacking hemoglobin β-cysteine93 S-nitrosylation

Systemic hypoxia is characterized by peripheral vasodilation and pulmonary vasoconstriction. However, the system-wide mechanism for signaling hypoxia remains unknown. Accumulating evidence suggests that hemoglobin (Hb) in RBCs may serve as an O2 sensor and O2-responsive NO signal transducer to regulate systemic and pulmonary vascular tone, but this remains unexamined at the integrated system level. One residue invariant in mammalian Hbs, β-globin cysteine93 (βCys93), carries NO as vasorelaxant S-nitrosothiol (SNO) to autoregulate blood flow during O2 delivery. βCys93Ala mutant mice thus exhibit systemic hypoxia despite transporting O2 normally. Here, we show that βCys93Ala mutant mice had reduced S-nitrosohemoglobin (SNO-Hb) at baseline and upon targeted SNO repletion and that hypoxic vasodilation by RBCs was impaired in vitro and in vivo, recapitulating hypoxic pathophysiology. Notably, βCys93Ala mutant mice showed marked impairment of hypoxic peripheral vasodilation and developed signs of pulmonary hypertension with age. Mutant mice also died prematurely with cor pulmonale (pulmonary hypertension with right ventricular dysfunction) when living under low O2. Altogether, we identify a major role for RBC SNO in clinically relevant vasodilatory responses attributed previously to endothelial NO. We conclude that SNO-Hb transduces the integrated, system-wide response to hypoxia in the mammalian respiratory cycle, expanding a core physiological principle.

Essential Role of Hemoglobin βCys93 in Cardiovascular Physiology.

The supply of oxygen to tissues is controlled by microcirculatory blood flow. One of the more surprising discoveries in cardiovascular physiology is the critical dependence of microcirculatory blood flow on a single conserved cysteine within the β-subunit (βCys93) of hemoglobin (Hb). βCys93 is the primary site of Hb S-nitrosylation [i.e., S-nitrosothiol (SNO) formation to produce S-nitrosohemoglobin (SNO-Hb)]. Notably, S-nitrosylation of βCys93 by NO is favored in the oxygenated conformation of Hb, and deoxygenated Hb releases SNO from βCys93. Since SNOs are vasodilatory, this mechanism provides a physiological basis for how tissue hypoxia increases microcirculatory blood flow (hypoxic autoregulation of blood flow). Mice expressing βCys93A mutant Hb (C93A) have been applied to understand the role of βCys93, and RBCs more generally, in cardiovascular physiology. Notably, C93A mice are unable to effect hypoxic autoregulation of blood flow and exhibit widespread tissue hypoxia. Moreover, reactive hyperemia (augmentation of blood flow following transient ischemia) is markedly impaired. C93A mice display multiple compensations to preserve RBC vasodilation and overcome tissue hypoxia, including shifting SNOs to other thiols on adult and fetal Hbs and elsewhere in RBCs, and growing new blood vessels. However, compensatory vasodilation in C93A mice is uncoupled from hypoxic control, both peripherally (e.g., predisposing to ischemic injury) and centrally (e.g., impairing hypoxic drive to breathe). Altogether, physiological studies utilizing C93A mice are confirming the allosterically controlled role of SNO-Hb in microvascular blood flow, uncovering essential roles for RBC-mediated vasodilation in cardiovascular physiology and revealing new roles for RBCs in cardiovascular disease.

Abstract 95: S-nitrosylation Therapy To Improve Ex-vivo Parameters Of Vascular Composite (vca): Study On Brain Dead Porcine

S-Nitrosylation Improves ex-vivo parameters of vascular composite (VCA): Study on brain dead swine Background: Brain death (BD) results in disruption of S-Nitrosohemoglobin (SNO-Hb) leading to decrease tissue micro perfusion. In a pre-clinical study on bd swine, we found that repletion of SNO-Hb by S-nitrosylation agent as ethyl nitrite (ENO) improves organ function. Vascularized composite allograft (VCA) is a viable therapeutic option for correction of composite tissue loss as limb amputation, abdominal trauma and severe facial damage. Although significant progress has been made with VCA surgical techniques and anti-rejection drug regimens, less attention has been directed to care of the tissue composite preservation. It is the physiologic status of the donor that often drives tissue availability and short-term VCA outcome. We hypothesized that additional of ENO during composite storage could restore composite microcirculation and thus improves outcome. Methods: VCA (n=8) and hind limb (n=6) were harvested from induced bd swine. Each composite was placed on a Lifeport storage machine and cold pulsatile perfusion was started at a targeted pressure 22 ± 5 mmHg for VCA and 13±2 for limb. Flow and resistance measurements were recorded during the experiment. ENO was aerated to the preservative solution for 2 hours (50ppm) for the treated composites. Data points were extracted from machine-generated values. Rate of change (slope) in flow rate and resistance were calculated. We compared values from control group to ENO treated group using Wilcoxon rank sum tests. GraphPad Prism software was used and considered a p < 0.05 to be statistically significant. Results: Flow and resistance measurements were expressed as percent change from baseline. For the duration of the pumping period. VCA flow rate in the ENO-treated group (n=2) was significantly higher (p<0.0001; 95% CI -17.22 to -9.743) than the control group (n=6), As well as, resistance was significantly decreased in ENO-treated group (p=0.0004; 95% CI 11.86 to 33.96). Similarly, limbs flow rate in the ENO-treated group (n=3) was significantly higher (p<0.0001; 95% CI -11.52 to -2.36) than control limb (n=3) as well as resistance significantly decreased in ENO-treated group (p<0.0001; 95% CI 11.51 to 24.13). Conclusions: S-nitrosylation therapy (ENO) during ex vivo storage of VCA holds promise to increase the number and quality of VCA available for transplant.

Hemoglobin β93 Cysteine Is Not Required for Export of Nitric Oxide Bioactivity From the Red Blood Cell.

Nitrosation of a conserved cysteine residue at position 93 in the hemoglobin β chain (β93C) to form S-nitroso (SNO) hemoglobin (Hb) is claimed to be essential for export of nitric oxide (NO) bioactivity by the red blood cell (RBC) to mediate hypoxic vasodilation and cardioprotection. To test this hypothesis, we used RBCs from mice in which the β93 cysteine had been replaced with alanine (β93A) in a number of ex vivo and in vivo models suitable for studying export of NO bioactivity. In an ex vivo model of cardiac ischemia/reperfusion injury, perfusion of a mouse heart with control RBCs (β93C) pretreated with an arginase inhibitor to facilitate export of RBC NO bioactivity improved cardiac recovery after ischemia/reperfusion injury, and the response was similar with β93A RBCs. Next, when human platelets were coincubated with RBCs and then deoxygenated in the presence of nitrite, export of NO bioactivity was detected as inhibition of ADP-induced platelet activation. This effect was the same in β93C and β93A RBCs. Moreover, vascular reactivity was tested in rodent aortas in the presence of RBCs pretreated with S-nitrosocysteine or with hemolysates or purified Hb treated with authentic NO to form nitrosyl(FeII)-Hb, the proposed precursor of SNO-Hb. SNO-RBCs or NO-treated Hb induced vasorelaxation, with no differences between β93C and β93A RBCs. Finally, hypoxic microvascular vasodilation was studied in vivo with a murine dorsal skin-fold window model. Exposure to acute systemic hypoxia caused vasodilatation, and the response was similar in β93C and β93A mice. RBCs clearly have the fascinating ability to export NO bioactivity, but this occurs independently of SNO formation at the β93 cysteine of Hb.

Abstract 365: Potential Relationship Between Nitric Oxide Bioactivity And Kidney Function Following Cardio-pulmonary Bypass

Background: Impaired kidney function is frequently observed in heart surgery patients following cardio-pulmonary bypass. Previous studies by our group have linked acute reductions in nitric oxide bioactivity, specifically declines in the main regulator of tissue blood flow S-nitrosohemoglobin (SNO-Hb), to reductions in kidney oxygenation and blood flow and subsequent renal impairment. For the current study, we hypothesized that components of the bypass procedure (viz. transfusion and red blood cell trauma) would reduce circulating SNO-Hb levels with the magnitude of this reduction correlating with markers of kidney function and patient outcome. Methods: After obtaining written informed consent, blood samples were obtained before and during bypass and on post-operative day (POD) 1. In addition to quantifying SNO-Hb samples were analyzed for change in kidney function (estimated glomerular filtration rate; eGFR), and plasma erythropoietin (EPO) concentration. Status and outcome data were secured from the patients’ medical record. Results: In the patients studied to date (n=30), there was a significant decline in SNO-Hb concentration by POD 1, the level of which inversely correlated with ICU stay (R=-0.32). In addition, there was a direct correlation between SNO-Hb and eGFR on PODs 1 &amp; 2 (R=0.48 and 0.30, respectively), i.e. lower SNO-Hb tracked with reductions in kidney function. Consistent with a presumed reduction in kidney oxygenation, we observed a significant rise in plasma EPO levels when coming off pump that were sustained into POD 1. Furthermore, the rise in EPO was inversely correlated with both SNO-Hb (R=-0.53) and eGFR (R=-0.55). Conclusion: CPB decreases SNO-Hb and is associated with kidney dysfunction and worse patient outcome (e.g. increased ICU stay). While the mechanism for this decline remains to be determined, the results suggest that therapies directed at maintaining or increasing NO bioactivity could improve outcome in adult cardiac surgery patients.

S-Nitrosohemoglobin Levels and Patient Outcome After Transfusion During Pediatric Bypass Surgery.

Banked blood exhibits impairments in nitric oxide (NO)‐based oxygen delivery capability, reflected in rapid depletion of S‐nitrosohemoglobin (SNO‐Hb). We hypothesized that transfusion of even freshly‐stored blood used in pediatric heart surgery would reduce SNO‐Hb levels and worsen outcome. In a retrospective review (n = 29), the percent of estimated blood volume (% eBV) replaced by transfusion directly correlated with ventilator time and inversely correlated with kidney function; similar results were obtained in a prospective arm (n = 20). In addition, an inverse association was identified between SNO‐Hb and postoperative increase in Hb (∆Hb), reflecting the amount of blood retained by the patient. Both SNO‐Hb and ∆Hb correlated with the probability of kidney dysfunction and oxygenation‐related complications. Further, regression analysis identified SNO‐Hb as an inverse predictor of outcome. The findings suggest that SNO‐Hb and ∆Hb are prognostic biomarkers following pediatric cardiopulmonary bypass, and that maintenance of red blood cell‐derived NO bioactivity might confer therapeutic benefit.

Renitrosylation of banked human red blood cells improves deformability and reduces adhesivity.

Transfusion of red blood cells (RBCs) is a frequent health care practice. However, unfavorable consequences may occur from transfusions of stored RBCs and are associated with RBC changes during storage. Loss of S-nitrosohemoglobin (SNO-Hb) and other S-nitrosothiols (SNOs) during storage is implicated as a detriment to transfusion efficacy. It was hypothesized that restoring SNOs within banked RBCs would improve RBC functions relevant to successful transfusion outcomes, namely, increased deformability and decreased adhesivity. Stored human RBCs were incubated with nitric oxide (NO) donors PROLI/NO and DEA/NO (disodium 1-[2-(carboxylato)-pyrrolidin-1-yl]diazen-1-ium-1,2-diolate and diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate) under varying experimental conditions (e.g., aerobic/anaerobic incubation, NO donor to RBC ratio). SNO restoration was evaluated in vitro and in vivo as a means to improve RBC function after storage. Incubation of RBCs with the NO donors resulted in 10-fold greater levels of SNO-Hb versus untreated control or sham RBCs, with significantly higher Hb-bound NO yields from an NO dose delivered by DEA/NO. RBC incubation with DEA/NO at a stoichiometry of 1:62.5 NO:Hb significantly increased RBC deformabilty and reduced adhesion to cultured endothelial cells. RBC incubation with DEA/NO also increased S-nitrosylation of RBC cytoskeletal and membrane proteins, including the β-spectrin chain. Renitrosylation attenuated both RBC sequestration in the lung and the mild blood oxygen saturation impairments seen with banked RBCs in a mouse model of transfusion. RBC renitrosylation using NO donors has promise for correcting deficient properties (e.g., adhesivity, rigidity, and SNO loss) of banked RBCs and in turn improving transfusion outcomes.

Chromatographic Separation Of Fetal and Adult Hemoglobins For Nitric Oxide Quantification In Term Human Umbilical Cord Blood

IntroductionNitric oxide (NO) is a vasoactive molecule that can bind to hemoglobin (Hb) in the form of S-nitrosothiol (SNO) functionalities at the β93 cysteine residues. Red blood cells (RBCs) containing S-nitrosohemoglobin (SNO-Hb) are able to not only deliver oxygen, but release vasodilatory NO/SNO equivalents to enhance blood flow in order to match tissue oxygen demand (e.g., during hypoxic vasodilation). Newborn babies normally carry two variants of hemoglobin, adult (Hb A) and fetal (Hb F). It is well known that Hb F binds oxygen more tightly than Hb A in order to facilitate oxygen scavenging across the placenta from maternal Hb A. Given that SNO-Hb is preferentially formed on oxygenated Hb, we hypothesized that Hb F may also bind a higher concentration of NO. Previous studies examining the NO content of cord blood were disadvantaged by looking at cord blood Hb as a whole. To date, no attempt has been made to determine the basal levels of NO bound to each Hb variant independently. Therefore, we aimed to separate the variants and measure the NO/SNO content of Hbs F and A in order to establish basal levels for each variant in cord blood at term. We reasoned that the results could improve insight into mechanisms of abnormal perinatal transitions and the selection of therapies involving NO signaling and/or RBC transfusion. MethodsVenous and arterial umbilical cord blood samples were collected immediately after normal term cesarean sections of infants with minimum gestational age of 37 weeks. RBC samples were washed in pH 7.4 phosphate buffered saline (PBS) with 100 µM diethylene triamine pentaacetic acid (DTPA) chelator to preserve SNOs, and hypotonically lysed. Total Hb was obtained through purification of the lysate through a Sephadex G-25 column. Partially purified Hb (200 µL) was loaded onto a HiTrap Q HP anionic exchange column (5 mL column volume with 34 µm bead size) and subjected to an increasing ionic strength gradient of 0–0.15M NaCl in pH 8.4 Tris buffer. Spectrophotometric analysis corroborated the complete separation of the variants. Isoelectric focusing on a Perkin Elmer Hemoglobin Resolve gel for 50 minutes at 1500 V and 10–15 °C was used in conjunction with an AFSC Hemopure control to identify the respective variants in each fraction. Each Hb variant was reconcentrated in pH 7.4 PBS with 100 µM DTPA via centrifugation through pre-rinsed 10 kDa MW cutoff centrifugal filters. The SNO/NO content of each variant was analyzed by photolysis-chemiluminescence of paired samples diluted to 100 μM Hb with/without 600 μM HgCl2. For samples with lower Hb concentration, a 6-fold molar excess of HgCl2 was also used. The Hg (mercury) acts to cleave NO bound to thiols (i.e., SNO) and is unreactive towards FeNO complexes; thus the difference in the paired sample peaks indicates the amount of SNO-Hb present in the sample. ResultsIn arterial cord blood samples, the amount of SNO-Hb bound to each variant (i.e., fetal and adult) was found to average ∼5 x 10-4 mol SNO per mol of Hb tetramer (Figure 1A). There was no significant difference between Hb F and Hb A with regards to SNO-Hb. Venous cord blood samples had similar results. The amount of total NO (i.e., SNO-Hb and heme-bound NO) bound to each variant was significantly higher on Hb A in arterial samples. (Figure 1B, *, p value < 0.05 vs. Hb F by paired t-test). [Display omitted] ConclusionsBoth Hb A and Hb F carry substantial and similar amounts of SNO adduct. Given the lower percentage (∼15-20%) of Hb A as a constituent in the total Hb of cord blood at term, the finding of a higher total NO content on Hb A than Hb F suggests that the presence of Hb A may be important to the total NO bioavailability in newborns. Vasodilator NO/SNO is known to be essential in the healthy cardiopulmonary transition to air breathing at birth. Thus, fundamental and translational studies assessing these species in newborns experiencing difficult transitions or pathophysiological states (e.g., persistent pulmonary hypertension of the newborn, PPHN) may provide potential biomarkers with utility in early detection of disease, prognosis, and intervention selection and management. The results and methodology presented here have broad applications to numerous areas of hematology and other medicine including neonatal intensive care, therapeutic induction of HbF in sickle cell disease patients, and decision-making in transfusion medicine. Disclosures:No relevant conflicts of interest to declare.

S-nitrosylation therapy to improve oxygen delivery of banked blood

From the perspectives of disease transmission and sterility maintenance, the world's blood supplies are generally safe. However, in multiple clinical settings, red blood cell (RBC) transfusions are associated with adverse cardiovascular events and multiorgan injury. Because ∼85 million units of blood are administered worldwide each year, transfusion-related morbidity and mortality is a major public health concern. Blood undergoes multiple biochemical changes during storage, but the relevance of these changes to unfavorable outcomes is unclear. Banked blood shows reduced levels of S-nitrosohemoglobin (SNO-Hb), which in turn impairs the ability of stored RBCs to effect hypoxic vasodilation. We therefore reasoned that transfusion of SNO-Hb-deficient blood may exacerbate, rather than correct, impairments in tissue oxygenation, and that restoration of SNO-Hb levels would improve transfusion efficacy. Notably in mice, administration of banked RBCs decreased skeletal muscle pO2, but infusion of renitrosylated cells maintained tissue oxygenation. In rats, hemorrhage-induced reductions in muscle pO2 were corrected by SNO-Hb-repleted RBCs, but not by control, stored RBCs. In anemic awake sheep, stored renitrosylated, but not control RBCs, produced sustained improvements in O2 delivery; in anesthetized sheep, decrements in hemodynamic status, renal blood flow, and kidney function incurred following transfusion of banked blood were also prevented by renitrosylation. Collectively, our findings lend support to the idea that transfusions may be causally linked to ischemic events and suggest a simple approach to prevention (i.e., SNO-Hb repletion). If these data are replicated in clinical trials, renitrosylation therapy could have significant therapeutic impact on the care of millions of patients.

Oral supplementation with glycine reduces oxidative stress in patients with metabolic syndrome, improving their systolic blood pressure

Reactive oxygen species derived from abdominal fat and uncontrolled glucose metabolism are contributing factors to both oxidative stress and the development of metabolic syndrome (MetS). This study was designed to evaluate the effects of daily administration of an oral glycine supplement on antioxidant enzymes and lipid peroxidation in MetS patients. The study included 60 volunteers: 30 individuals that were supplemented with glycine (15 g/day) and 30 that were given a placebo for 3 months. We analysed thiobarbituric acid reactive substances (TBARS) and S-nitrosohemoglobin (SNO-Hb) in plasma; the enzymatic activities of glucose-6-phosphate dehydrogenase (G6PD), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) in erythrocytes; and the expression of CAT, GPX, and SOD2 in leukocytes. Individuals treated with glycine showed a 25% decrease in TBARS compared with the placebo-treated group. Furthermore, there was a 20% reduction in SOD-specific activity in the glycine-treated group, which correlated with SOD2 expression. G6PD activity and SNO-Hb levels increased in the glycine-treated male group. Systolic blood pressure (SBP) also showed a significant decrease in the glycine-treated men (p = 0.043). Glycine plays an important role in balancing the redox reactions in the human body, thus protecting against oxidative damage in MetS patients.

Endocrine Nitric Oxide Bioactivity and Hypoxic Vasodilation by Inhaled Nitric Oxide

Inhaled nitric oxide (iNO) is approved by the Food and Drug Administration for the treatment of persistent pulmonary hypertension of the newborn, largely as a result of the pioneering research efforts of Warren Zapol. Inhaled nitric oxide (NO) dilates pulmonary resistance vessels to improve ventilation–perfusion matching before being inactivated by reactions with hemoglobin (Hb) in blood exiting the lung. Inhaled NO therefore is a selective pulmonary vasodilator.1 Notwithstanding the validity of this model, investigators including Claes Frostell2 and David Wessel3 noted early on that high therapeutic doses of iNO led to subtle decreases in blood pressure. It had recently been discovered that albumin could be S-nitrosylated by NO,4 endowing it with long-lived vasodilatory activity. So, when presented with the quandary of systemic effects, it was quickly demonstrated that iNO could S-nitrosylate plasma proteins,5 potentially providing systemic vasodilatory activity6 and thus acting in an endocrine-like manner. S-nitrosoalbumin since has been implicated in protection by iNO against reperfusion injury in systemic vessels,7,8 but also may form within airways of iNO-treated patients,5,9 potentially contributing to “pulmonary selectivity.” In subsequent studies, the family of circulating S-nitrosylated proteins (SNO-proteins) was expanded to include S-nitrosohemoglobin (SNO-Hb),10 which exhibits the remarkable ability to mediate hypoxic vasodilation,11 the selective vasodilation in proportion to degree of hypoxemia. A number of recent studies have provided evidence that iNO may utilize SNO-based pathways12 to confer protection against ischemic insults8,13–18—none more remarkable than the report by Terpolilli et al19 in this issue of Circulation Research , which illustrates beautifully in cerebral vessels the principles of endocrine NO bioactivity and hypoxia-coupled delivery of NO bioactivity. Their demonstration that iNO promotes dilation of cerebral resistance vessels selectively in hypoperfused tissue, without changes …

Erythrocyte-dependent regulation of human skeletal muscle blood flow: role of varied oxyhemoglobin and exercise on nitrite, S-nitrosohemoglobin, and ATP

The erythrocyte is proposed to play a key role in the control of local tissue perfusion via three O(2)-dependent signaling mechanisms: 1) reduction of circulating nitrite to vasoactive NO, 2) S-nitrosohemoglobin (SNO-Hb)-dependent vasodilatation, and 3) release of the vasodilator and sympatholytic ATP; however, their relative roles in vivo remain unclear. Here we evaluated each mechanism to gain insight into their roles in the regulation of human skeletal muscle blood flow during hypoxia and hyperoxia at rest and during exercise. Arterial and femoral venous hemoglobin O(2) saturation (O(2)Hb), plasma and erythrocyte NO and ATP metabolites, and leg and systemic hemodynamics were measured in 10 healthy males exposed to graded hypoxia, normoxia, and graded hyperoxia both at rest and during submaximal one-legged knee-extensor exercise. At rest, leg blood flow and NO and ATP metabolites in plasma and erythrocytes remained unchanged despite large alterations in O(2)Hb. During exercise, however, leg and systemic perfusion and vascular conductance increased in direct proportion to decreases in arterial and venous O(2)Hb (r(2) = 0.86-0.98; P = 0.01), decreases in venous plasma nitrite (r(2) = 0.93; P < 0.01), increases in venous erythrocyte nitroso species (r(2) = 0.74; P < 0.05), and to a lesser extent increases in erythrocyte SNO (r(2) = 0.59; P = 0.07). No relationship was observed with plasma ATP (r(2) = 0.01; P = 0.99) or its degradation compounds. These in vivo data indicate that, during low-intensity exercise and hypoxic stress, but not hypoxic stress alone, plasma nitrite consumption and formation of erythrocyte nitroso species are associated with limb vasodilatation and increased blood flow in the human skeletal muscle vasculature.

The red blood cell storage lesion: a method to the madness

The red blood cell storage lesion: a method to the madness

SNO-hemoglobin is not essential for red blood cell–dependent hypoxic vasodilation

The coupling of hemoglobin sensing of physiological oxygen gradients to stimulation of nitric oxide (NO) bioactivity is an established principle of hypoxic blood flow. One mechanism proposed to explain this oxygen-sensing-NO bioactivity linkage postulates an essential role for the conserved Cys93 residue of the hemoglobin beta-chain (betaCys93) and, specifically, for S-nitrosation of betaCys93 to form S-nitrosohemoglobin (SNO-Hb). The SNO-Hb hypothesis, which conceptually links hemoglobin and NO biology, has been debated intensely in recent years. This debate has precluded a consensus on physiological mechanisms and on assessment of the potential role of SNO-Hb in pathology. Here we describe new mouse models that exclusively express either human wild-type hemoglobin or human hemoglobin in which the betaCys93 residue is replaced with alanine to assess the role of SNO-Hb in red blood cell-mediated hypoxic vasodilation. Substitution of this residue, precluding hemoglobin S-nitrosation, did not change total red blood cell S-nitrosothiol abundance but did shift S-nitrosothiol distribution to lower molecular weight species, consistent with the loss of SNO-Hb. Loss of betaCys93 resulted in no deficits in systemic or pulmonary hemodynamics under basal conditions and, notably, did not affect isolated red blood cell-dependent hypoxic vasodilation. These results demonstrate that SNO-Hb is not essential for the physiologic coupling of erythrocyte deoxygenation with increased NO bioactivity in vivo.

Restoring Oxygen Carrying Capacity of Sickle Erythrocytes with Nitric Oxide Donors.

Sickle cell anemia (SCA) is an inherited blood disorder of hemoglobin function. A genetic mutation results in the substitution of a valine for glutamic acid residue at position 6 of the beta-globin chain yielding the mutant hemoglobin S (HbS). HbS polymerizes within erythrocytes during deoxygenation resulting in altered affinity of oxygen binding. The slightly different P50 (PO2 at which Hb is half-saturated with oxygen) values of sickle erythrocytes obtained during either oxygenation or deoxygenation (hysteresis) demonstrate HbS polymerization induced inhibition of oxygen affinity. Nitric oxide (NO) has been found to be an important signaling molecule in the circulatory system. NO derivatives of Hb provide insights into the physiological role of Hb. NO can bind to Hb at either the heme moiety forming nitrosylhemoglobin (HbNO) or to the conserved beta-93 cysteine yielding S-nitrosohemoglobin (SNO-Hb). In deoxygenated venous blood NO preferentially binds to the hemes of Hb forming HbNO while in oxygenated arterial blood NO binds to the beta-93 cysteine residues forming SNO-Hb. Increased oxygen affinity is seen in both SNO-Hb (Bonaventura C, et al, 1999) and also with HbNO. Decreasing the HbS P50 inhibits intra-erythrocyte HbS polymerization that may be an effective strategy to treat SCA. Clinical trials of NO breathing effects on oxygen affinity are conflicting. One study found an increased oxygen affinity of blood from SCA patients breathing 80 ppm NO with no effect seen in normal controls (Head A, et al, 1997). Another study found that levels of NO bound to Hb are too low to affect overall oxygen affinity (Gladwin M, et al, 1999). The purpose of this in vitro study was to determine the oxygen affinity of deoxygenated sickle erythrocytes pre-treated with exogenous NO donors. Blood from SCA (HbSS) and normal controls (HbAA) were collected and suspended in PBS buffer and deoxygenated with argon gas. The Hb concentration of each sample was calculated and then was either left untreated (control) or treated with varying concentrations of NO donors. The NO donors included: 2-(N, N-diethylamino)-diazenolate-2-oxide (DEANO), S-nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside (SNP), an aqueous solution of NO, and sodium trioxodinitrate (Angeli's salt, AS). Methemoglobin and protein degradation were negligible. Samples were then transferred via airtight syringes into a stirred and temperature controlled (37°C) chamber of PBS solution at ambient oxygen pressure fitted with a very sensitive oxygen electrode. Oxygen levels were measured in real time. The amount of oxygen extracted from the PBS medium followed first order kinetics. Studies with HbSS red cell suspensions showed that the largest increment in oxygen extraction from the medium was obtained with DEANO pre-treatment. Calculations indicated that low levels of NO treatment, at approximately a 1:1000 ratio of [NO]/[heme], yielded the largest oxygen consumption. The effects of pre-treatment with the other NO donors on sickle erythrocytes (HbSS) were not as pronounced. DEANO is an NO donor yielding a “pure” NO radical as opposed to other redox forms. Similar studies with HbAA and HbSC did not show increases in oxygen extraction. Taken together the data suggest that low levels of NO perturb the quaternary structure of intraerythrocyte HbS polymer allowing depolymerization and increased oxygen affinity. The hope is that these in vitro studies will better characterize the role of NO in its interactions with Hb and the red cell and to use this knowledge for potential therapies in diseases such as SCA.

S-nitrosohemoglobin deficiency: a mechanism for loss of physiological activity in banked blood.

RBCs distribute oxygen to tissues, but, paradoxically, blood transfusion does not always improve oxygen delivery and is associated with ischemic events. We hypothesized that storage of blood would result in loss of NO bioactivity, impairing RBC vasodilation and thus compromising blood flow, and that repleting NO bioactivity would restore RBC function. We report that S-nitrosohemoglobin (SNO-Hb) concentrations declined rapidly after storage of fresh venous blood and that hypoxic vasodilation by banked RBCs correlated strongly with the amounts of SNO-Hb (r(2) = 0.90; P < 0.0005). Renitrosylation of banked blood during storage increased the SNO-Hb content and restored its vasodilatory activity. In addition, canine coronary blood flow was greater during infusion of renitrosylated RBCs than during infusion of S-nitrosothiol-depleted RBCs, and this difference in coronary flow was accentuated by hypoxemia (P < 0.001). Our findings indicate that NO bioactivity is depleted in banked blood, impairing the vasodilatory response to hypoxia, and they suggest that SNO-Hb repletion may improve transfusion efficacy.

Vascular Smooth Muscle NO Exposure from Intraerythrocytic SNOHb: A Mathematical Model

We previously constructed computational models based on the biochemical pathway analysis of different nitric oxide (NO) synthase isoforms and found a large discrepancy between our predictions and perivascular NO measurements, suggesting the existence of nonenzymatic sources of NO. S-nitrosohemoglobin (SNOHb) has been suggested as a major source to release NO in the arteriolar lumen and induce hypoxic vasodilation. In the present study, we formulated a multicellular computational model to quantify NO exposure in arteriolar smooth muscle when the NO released by intraerythrocytic SNOHb is the sole NO source in the vasculature. Our calculations show an NO exposure of approximately 0.25-6 pM in the smooth muscle region. This amount does not account for the large discrepancy we encountered regarding perivascular NO levels. We also found that the amount of NO delivered by SNOHb to smooth muscle strongly depends on the SNOHb concentration and half-life, which further determine the rate of NO release, as well as on the membrane permeability of red blood cells (RBCs) to NO. In conclusion, our mathematical model predicts that picomolar amounts of NO can be delivered to the vascular smooth muscle by intraerythrocytic SNOHb; this amount of NO alone appears not sufficient to induce the hypoxic vasodilation.