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Long-term consumption of beetroot juice on efficacy of converting dietary nitrate to plasma nitrate and nitrite was investigated. Adults were randomized to consume either beetroot juice with 380 mg of nitrate (BR) or a beetroot juice placebo (PL) for 12-weeks. Plasma nitrate and nitrite were measured before and 90-minutes after consuming their intervention beverage. Percent change in nitrite across the 90 min was greater in BR (273.2 ± 39.9%) vs. PL (4.9 ± 36.9%). Long-term consumption of nitrate containing beetroot juice increased fasting nitrate and nitrite plasma levels compared to baseline.

Malaria is a highly oxidative parasitic disease in which anemia is the most common clinical symptom. A major contributor to malarial anemia pathogenesis is the destruction of bystander, uninfected red blood cells. Metabolic fluctuations are known to occur in the plasma of individuals with acute malaria, emphasizing the role of metabolic changes in disease progression and severity. Here, we report that conditioned media fromPlasmodium falciparumculture induces oxidative stress in healthy uninfected RBCs. Additionally, we show the benefit of amino acid pre-exposure for RBCs and how this pre-treatment intrinsically prepares RBCs to mitigate oxidative stress.Key pointsIntracellular ROS is acquired in red blood cells incubated withPlasmodium falciparumconditioned mediaGlutamine, cysteine, and glycine amino acid supplementation increased glutathione biosynthesis and reduced ROS levels in stressed RBCs

Ex vivo labeling withAutologous units of whole blood were divided equally into two bags and stored under standard blood bank conditions at 2 to 6°C (N = 4 healthy adult volunteers). One bag was biotinylated (15 μg/ml) on storage days 5 to 7 (fresh) and the other was biotinylated (3 μg/ml) on days 35 to 42 (aged). The proportion of circulating BioRBC was measured serially, and cell-surface biotin was quantified with reference to molecules of equivalent soluble fluorochrome. Clearance kinetics were modeled by RBC age distribution at infusion (Gaussian vs. uniform) and decay over time (constant vs. exponential).Data were consistent with biphasic exponential clearance of cells of uniform age. Our best estimate of BioRBC clearance (half-life [TSimilar clearance kinetics of fresh and aged BioRBC may be due to the extensive washing required during biotinylation. Survival kinetics consistent with cells with uniform rather than Gaussian or other non-uniform age distributions suggest that washing, and potentially RBC culling, may extend the storage life of RBC products.

Nitric oxide, NO, has been explored as a therapeutic agent to treat thrombosis. In particular, NO has potential in treating mechanical device-associated thrombosis due to its ability to reduce platelet activation and due to the central role of platelet activation and adhesion in device thrombosis. Nitrite is a unique NO donor that reduces platelet activation in that it’s activity requires the presence of red blood cells whereas NO activity of other NO donors is blunted by red blood cells. Interestingly, we have previously shown that red blood cell mediated inhibition of platelet activation by adenosine diphosophate (ADP) is dramatically enhanced by illumination with far-red light that is likely due to photolysis of red cell surface bound NO congeners. We now report the effects of nitrite, far-red light, and their combination on several measure of blood coagulation using a variety of agonists. We employed turbidity assays in platelet rich plasma, platelet activation using flow cytometry analysis of a fluorescently labeled antibody to the activated platelet fibrinogen binding site, multiplate impedance-based platelet aggregometry, and assessment of platelet adhesion to collagen coated flow-through microslides. In all cases, the combination of far-red light and nitrite treatment decreased measures of coagulation, but in some cases mono-treatment with nitrite or light alone had no effect. Perhaps most relevant to device thrombosis, we observed that platelet adhesions was inhibited by the combination of nitrite and light treatment while nitrite alone and far-red light alone trended to decrease adhesion, but the results were mixed. These results support the potential of combined far-red light and nitrite treatment for preventing thrombosis in extra-corporeal or shallow-tissue depth devices where the far-red light can penetrate. Such a combined treatment could be advantageous due to the localized treatment afforded by far-red light illumination with minimal systemic effects. Given the role of thrombosis in COVID 19, application to treatment of patients infected with SARS Cov-2 might also be considered.

Beetroot juice is a food high in nitrate and is associated with cardiometabolic health benefits and enhanced exercise performance through the production of nitric oxide in the nitrate–nitrite–nitric oxide pathway. Since various food components influence this pathway, the aim of this trial was to study the effect of beetroot juice alone and in conjunction with vitamin C or protein on the acute response to plasma nitrate and nitrite levels in healthy middle- to older-aged adults. In this cross-over trial, each participant received, in a randomized order, a single dose of Beet It Sport® alone; Beet It Sport®, plus a 200 mg vitamin C supplement; and Beet It Sport® plus 15 g of whey protein. Plasma levels of nitrate and nitrite were determined prior to and at 1 and 3 h after intervention. Log plasma nitrate and nitrite was calculated to obtain data that were normally distributed, and these data were analyzed using two-way within-factors ANOVA, with time and treatment as the independent factors. There were no statistically significant differences for log plasma nitrate (p = 0.308) or log plasma nitrite (p = 0.391) values across treatments. Log plasma nitrate increased significantly from pre-consumption levels after 1 h (p < 0.001) and 3 h (p < 0.001), but plasma nitrate was lower at 3 h than 1 h (p < 0.001). Log plasma nitrite increased from pre to 1 h (p < 0.001) and 3 h (p < 0.001) with log values at 3 h higher than at 1 h (p = 0.003). In this cohort, we observed no differences in log plasma nitrate and nitrite at 1 h and 3 h after co-ingesting beetroot juice with vitamin C or a whey protein supplement compared to beetroot juice alone. Further research needs to be undertaken to expand the blood-sampling time-frame and to examine factors that may influence the kinetics of the plasma nitrate to nitrite efficacy, such as differences in fluid volume and osmolarity between treatments employed.

Exercise tolerance appears to benefit most from dietary nitrate [Formula: see text] supplementation when muscle oxygen (O(2)) availability is low. Using a double-blind, randomized cross-over design, we tested the hypothesis that acute [Formula: see text] supplementation would improve blood flow restricted exercise duration in post-menopausal women, a population with reduced endogenous nitric oxide bioavailability. Thirteen women (57–76 yr) performed rhythmic isometric handgrip contractions (10% MVC, 30 per min) during progressive forearm blood flow restriction (upper arm cuff gradually inflated 20 mmHg each min) on three study visits, with 7 to 10 days between visits. Approximately one week following the first (familiarization) visit, participants consumed 140 ml of [Formula: see text] concentrated (9.7 mmol, 0.6 gm [Formula: see text]) or [Formula: see text] depleted beetroot juice (placebo) on separate days (≥ 7 days apart), with handgrip exercise beginning 100 min post-consumption. Handgrip force recordings were analyzed to determine if [Formula: see text] supplementation enhanced force development as blood flow restriction progressed. Nitrate supplementation increased plasma [Formula: see text] (16.2-fold) and [Formula: see text] (4.2-fold) and time to volitional fatigue (61.8±56.5 sec longer duration vs. placebo visit; p=0.003). Nitrate supplementation increased the rate of force development as forearm muscle ischemia progressed (p= 0.023 between 50 and 75% of time to fatigue) with non-significant effects thereafter (p=0.052). No effects of nitrate supplementation were observed for mean duration of contraction or relaxation rates (all p>0.150). These results suggest that acute [Formula: see text] supplementation prolongs time-to-fatigue and speeds grip force development during progressive forearm muscle ischemia in postmenopausal women.

Nitric Oxide (NO) is an important signaling molecule that plays roles in controlling vascular tone, hemostasis, host defense, and many other physiological functions. Low NO bioavailability contributes to pathology and NO administration has therapeutic potential in a variety of diseases. Thus, accurate measurements of NO bioavailability and reactivity are critical. Due to its short lifetime in vivo and many in vitro conditions, NO bioavailability and reactivity are often best determined by measuring NO congeners and metabolites that are more stable. Chemiluminescence-based detection of NO following chemical reduction of these compounds using the tri-iodide and vanadium chloride methods have been widely used in a variety of clinical and laboratory studies. In this review, we describe these methods used to detect nitrite, nitrate, nitrosothiols and other species and discuss limitations and proper controls.

I.v. administration of a high-affinity carbon monoxide-binding (CO-binding) molecule, recombinant neuroglobin, can improve survival in CO poisoning mouse models. The current study aims to discover how biochemical variables of the scavenger determine the CO removal from the RBCs by evaluating 3 readily available hemoproteins, 2,3-diphosphoglycerate stripped human hemoglobin (StHb); N-ethylmaleimide modified hemoglobin (NEMHb); and equine myoglobin (Mb). These molecules efficiently sequester CO from hemoglobin in erythrocytes in vitro. A kinetic model was developed to predict the CO binding efficacy for hemoproteins, based on their measured in vitro oxygen and CO binding affinities, suggesting that the therapeutic efficacy of hemoproteins for CO poisoning relates to a high M value, which is the binding affinity for CO relative to oxygen (KA,CO/KA,O2). In a lethal CO poisoning mouse model, StHb, NEMHb, and Mb improved survival by 100%, 100%, and 60%, respectively, compared with saline controls and were well tolerated in 48-hour toxicology assessments. In conclusion, both StHb and NEMHb have high CO binding affinities and M values, and they scavenge CO efficiently in vitro and in vivo, highlighting their therapeutic potential for point-of-care antidotal therapy of CO poisoning.

Altered mitochondrial function occurs in sickle cell disease (SCD), due in part to low nitric oxide (NO) bioavailability. Arginine, the substrate for NO production, becomes acutely deficient in SCD patients with vaso-occlusive pain episodes (VOE). To determine if arginine improves mitochondrial function, 12 children with SCD-VOE (13.6 ± 3 years; 67% male; 75% hemoglobin-SS) were randomized to 1 of 3 arginine doses: (1) 100 mg/kg IV 3 times/day (TID); (2) loading dose (200 mg/kg) then 100 mg/kg TID; or (3) loading dose (200 mg/kg) followed by continuous infusion (300 mg/kg per day) until discharge. Platelet-rich plasma mitochondrial activity, protein expression, and protein-carbonyls were measured from emergency department (ED) presentation vs discharge. All VOE subjects at ED presentation had significantly decreased complex-V activity compared to a steady-state cohort. Notably, complex-V activity was increased at discharge in subjects from all 3 arginine-dosing schemes; greatest increase occurred with a loading dose (P < .001). Although complex-IV and citrate synthase activities were similar in VOE platelets vs steady state, enzyme activities were significantly increased in VOE subjects after arginine-loading dose treatment. Arginine also decreased protein-carbonyl levels across all treatment doses (P < .01), suggesting a decrease in oxidative stress. Arginine therapy increases mitochondrial activity and reduces oxidative stress in children with SCD/VOE. This trial was registered at www.clinicaltrials.gov as #NCT02536170.

Background and purpose Although it has been reported that bovine carbonic anhydrase CAII is capable of generating NO from nitrite, the function and mechanism of CAII in nitrite-dependent NO formation and vascular responses remain controversial. We tested the hypothesis that CAII catalyses NO formation from nitrite and contributes to nitrite-dependent inhibition of platelet activation and vasodilation. Experiment approach The role of CAII in enzymatic NO generation was investigated by measuring NO formation from the reaction of isolated human and bovine CAII with nitrite using NO photolysis-chemiluminescence. A CAII-deficient mouse model was used to determine the role of CAII in red blood cell mediated nitrite reduction and vasodilation. Key results We found that the commercially available purified bovine CAII exhibited limited and non-enzymatic NO-generating reactivity in the presence of nitrite with or without addition of the CA inhibitor dorzolamide; the NO formation was eliminated with purification of the enzyme. There was no significant detectable NO production from the reaction of nitrite with recombinant human CAII. Using a CAII-deficient mouse model, there were no measurable changes in nitrite-dependent vasodilation in isolated aorta rings and in vivo in CAII-/- , CAII+/- , and wild-type mice. Moreover, deletion of the CAII gene in mice did not block nitrite reduction by red blood cells and the nitrite-NO-dependent inhibition of platelet activation. Conclusion and implications These studies suggest that human, bovine and mouse CAII are not responsible for nitrite-dependent NO formation in red blood cells, aorta, or the systemic circulation.

Background: 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. Methods: 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. Results: 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. Conclusions: RBCs clearly have the fascinating ability to export NO bioactivity, but this occurs independently of SNO formation at the β93 cysteine of Hb.

PURPOSE: Consumption of nitrate-rich beetroot juice can lower blood pressure in peripheral as well as central arteries and may exert additional hemodynamic benefits (e.g. reduced aortic wave reflections). The specific influence of nitrate supplementation on arterial pressures and aortic wave properties in postmenopausal women, a group that experiences accelerated increases in these variables with age, is unknown. Accordingly, the primary aim of this study was to determine the effect of consuming nitrate-rich beetroot juice on resting brachial and aortic blood pressures (BP) and pulse wave characteristics in a group of healthy postmenopausal women, in comparison to a true (nitrate-free beetroot juice) placebo. METHODS: Brachial (oscillometric cuff) and radial (SphygmoCor) pressures and derived-aortic waveforms were measured during supine rest in thirteen healthy postmenopausal women (63 ± 1 yr) before and 100 min after consumption of 140 ml of either nitrate-rich (9.7 mmol, 0.6 gm NO(3)(−)) or nitrate-depleted beetroot juice on randomized visits approximately 10 days apart (cross-over design). Ten young premenopausal women (22 ± 1 yr) served as a reference (non-supplemented) cohort. RESULTS: Brachial and derived-aortic variables showed the expected age-associated differences in these women (all p

OBJECTIVE: Red blood cell (RBC) hemolysis is one of the most common storage lesions in packed RBCs (pRBC). Older units of pRBCs, especially those >21 days old, have increasing levels of hemolysis leading to increased oxidative stress and premature platelet activation. This effect can mostly be attributed to the increase of cell-free hemoglobin (Hb). Therefore, removal of cell-free Hb from pRBCs prior to transfusion could mitigate these deleterious effects. We propose a new method for the removal of Hb from pRBCs using zinc beads. APPROACH AND RESULTS: Prepared Hb solutions and pRBCs were treated with zinc beads using two different protocols. UV-Vis spectrophotometry was used to determine Hb concentrations, before and after treatment. Experiments were run in triplicate and paired t-tests were used to determine significant differences between groups. Zinc beads removed on average 94% of cell-free Hb within 15 minutes and 78% Hb from pRBCs (p

Background Nitrite is reduced by heme-proteins and molybdenum-containing enzymes to form the important signaling molecule nitric oxide (NO), mediating NO signaling. Substantial evidence suggests that deoxygenated hemoglobin within red blood cells (RBCs) is the main erythrocytic protein responsible for mediating nitrite-dependent NO signaling. In other work, infrared and far red light have been shown to have therapeutic potential that some attribute to production of NO. Here we explore whether a combination of nitrite and far red light treatment has an additive effect in NO-dependent processes, and whether this effect is mediated by RBCs. Methods and results Using photoacoustic imaging in a rat model as a function of varying inspired oxygen, we found that far red light (660 nm, five min. exposure) and nitrite feeding (three weeks in drinking water at 100 mg/L) each separately increased tissue oxygenation and vessel diameter, and the combined treatment was additive. We also employed inhibition of human platelet activation measured by flow cytometry to assess RBC-dependent nitrite bioactivation and found that far red light dramatically potentiates platelet inhibition by nitrite. Blocking RBC-surface thiols abrogated these effects of nitrite and far-red light. RBC-dependent production of NO was also shown to be enhanced by far red light using a chemiluminescence-based nitric oxide analyzer. In addition, RBC-dependent bioactivation of nitrite led to prolonged lag times for clotting in platelet poor plasma that was enhanced by exposure to far red light. Conclusions Our results suggest that nitrite leads to the formation of a photolabile RBC surface thiol-bound species such as an S-nitrosothiol or heme-nitrosyl (NO-bound heme) for which far red light enhances NO signaling. These findings expand our understanding of RBC-mediated NO production from nitrite. This pathway of NO production may have therapeutic potential in several applications including thrombosis, and, thus, warrants further study., Graphical abstract Potential mechanism of RBC-mediated nitrite bioactivation and its potentiation by far red light. Nitrite reacts with deoxygenated Hb to make NO which then binds other vacant hemes or forms a nitrosothiol (RSNO). The nitrosyl-heme or nitrosothiol is exported and binds a surface thiol. Another potential NO species that may form is a DNIC (not shown). The NO congener can then be transported in plasma and interact with platelets and other blood components and this action is potentiated by photolysis using far red light.fx1, Highlights • Nitrite and far red light increase tissue oxygenation under hypoxia. • Far red light enhances nitrite-mediated inhibition of platelet activation by red cells. • Blocking red cell surface thiols abrogates nitrite-mediated effects. • NO production from nitrite and red cells is enhanced by far red light. • Lag time in clotting is prolonged by nitrite plus far red light.

Mycobacterium tuberculosis (Mtb) senses and responds to host-derived gasotransmitters NO and CO via heme-containing sensor kinases DosS and DosT and the response regulator DosR. Hydrogen sulfide (H2S) is an important signaling molecule in mammals, but its role in Mtb physiology is unclear. We have previously shown that exogenous H2S can modulate expression of genes in the Dos dormancy regulon via an unknown mechanism(s). Here, we tested the hypothesis that Mtb senses and responds to H2S via the DosS/T/R system. Using UV-Vis and EPR spectroscopy, we show that H2S binds directly to the ferric (Fe3+) heme of DosS (KD = 5.64 µM) but not the ferrous (Fe2+) form. No interaction with DosT was detected. Thus, the mechanism by which DosS senses H2S is different from that for sensing NO and CO, which bind only the ferrous forms of DosS and DosT. Steered Molecular Dynamics simulations show that H2S, and not the charged HS- species, can enter the DosS heme pocket. We also show that H2S increases DosS autokinase activity and subsequent phosphorylation of DosR, and H2S-mediated increases in Dos regulon gene expression is lost in Mtb lacking DosS. Finally, we demonstrate that physiological levels of H2S in macrophages can induce Dos regulon genes via DosS. Overall, these data reveal a novel mechanism whereby Mtb senses and responds to a third host gasotransmitter, H2S, via DosS-Fe3+. These findings highlight the remarkable plasticity of DosS and establish a new paradigm for how bacteria can sense multiple gasotransmitters through a single heme sensor kinase.Significance StatementHydrogen sulfide (H2S) is an important signaling molecule in eukaryotes and bacteria, and along with CO and NO, is an important part of host defense against Mycobacterium tuberculosis (Mtb). However, the mechanism(s) by which Mtb senses and responds to H2S is unknown. Here, we report that the Mtb heme sensor kinase DosS, a known sensor of CO and NO, is also a sensor of H2S. We found that H2S binds DosS in its ferric (Fe3+) state, which is considered as its inactive state, to induce the Dos dormancy regulon during infection. These data highlight the unusual capacity of Mtb to sense multiple gasotransmitters through a single sensing protein.

Cell-free hemoglobin (CFH) elevations are a known consequence of clinical sepsis. Using a two-by-two factorial design and extensive physiological and biochemical evidence, we found a direct mechanism of injury related to nitric oxide scavenging leading to pulmonary hypertension increasing right heart afterload, depressed cardiac function, worsening circulatory failure, and death, as well as an indirect mechanism related to iron toxicity. These discoveries alter conventional thinking about septic shock pathogenesis and provide novel therapeutic approaches.

Small artery vasodilation in response to hypoxia is essential for matching oxygen supply to tissue oxygen demand. One source of hypoxic dilation via nitric oxide (NO) signaling is nitrite reduction by erythrocytic hemoglobin (α2β2). However, the alpha subunit of hemoglobin is also expressed in resistance artery endothelium and localized to myoendothelial junctions, a subcellular domain that contacts underlying vascular smooth muscle cells. We hypothesized that nitrite reduction mediated by endothelial alpha globin may occur at myoendothelial junctions to regulate hypoxic vasodilation. To test this concept, we created two novel mouse strains: one lacking alpha globin specifically in endothelium (EC Hba1Δ/Δ) and one where alpha globin is mutated such that its inhibitory association with endothelial NO synthase (eNOS) is prevented (Hba1WT/Δ36-39). In EC Hba1Δ/Δ or Hba1WT/Δ36-39 mice hemoglobin levels, hematocrit and erythrocyte counts were unchanged from littermate controls. Loss of the full alpha globin protein from the endothelium in the EC Hba1Δ/Δ model was associated with decreased exercise capacity and decreased intracellular nitrite utilization in hypoxic conditions. These effects were not seen in Hba1WT/Δ36-39 animals. Hypoxia induced vasodilation was decreased by 60% in isolated thoracodorsal arteries from EC Hba1Δ/Δ, while infusion of erythrocytes only partially rescued the dilatory response. Lastly, unlike other models where blood pressure is decreased, EC Hba1Δ/Δ blood pressure was not altered in response to hypoxia. Overall, we conclude that alpha globin in the resistance artery endothelium can act as a nitrite reductase to provide a local vasodilatory response to hypoxia.

BackgroundHydroxychloroquine (HCQ) is widely used in the treatment of malaria, rheumatologic disease such as lupus, and most recently, COVID-19. These uses raise concerns about its safe use in the setting of glucose-6-phosphate dehydrogenase (G6PD) deficiency, especially as 11% of African American men carry the G6PD A- variant. However, limited data exist regarding the safety of HCQ in this population.Study design and methodsRecently, we created a novel "humanized" mouse model containing the G6PD deficiency A- variant (Val68Met) using CRISPR technology. We tested the effects of high-dose HCQ administration over 5 days on hemolysis in our novel G6PD A- mice. In addition to standard hematologic parameters including plasma hemoglobin, erythrocyte methemoglobin, and reticulocytes, hepatic and renal function were assessed after HCQ.ResultsResidual erythrocyte G6PD activity in G6PD A- mice was ~6% compared to wild-type (WT) littermates. Importantly, we found no evidence of clinically significant intravascular hemolysis, methemoglobinemia, or organ damage in response to high-dose HCQ.ConclusionsThough the effects of high doses over prolonged periods was not assessed, this study provides early, novel safety data of the use of HCQ in the setting of G6PD deficiency secondary to G6PD A-. In addition to novel safety data for HCQ, to our knowledge, we are the first to present the creation of a "humanized" murine model of G6PD deficiency.

Macrophages are main effectors of heme metabolism, increasing transiently in the liver during heightened disposal of damaged or senescent red blood cells (sRBC). Macrophages are also essential in defense against microbial threats, but pathologic states of heme excess may be immunosuppressive. Herein, we uncovered a mechanism whereby an acute rise in sRBC disposal by macrophages led to an immunosuppressive phenotype following intrapulmonary Klebsiella pneumoniae infection characterized by increased extrapulmonary bacterial proliferation and reduced survival from sepsis in mice. The impaired immunity to K. pneumoniae during heightened sRBC disposal was independent of iron acquisition by bacterial siderophores, as K. pneumoniae mutant lacking siderophore function recapitulated findings observed with wildtype strain. Rather, sRBC disposal induced a liver transcriptomic profile notable for suppression of Stat1 and interferon-related responses during K. pneumoniae sepsis. Excess heme handling by macrophages recapitulated STAT1 suppression during infection that required synergistic NRF1 and NRF2 activation but was independent of heme oxygenase-1 induction. Whereas iron was dispensable, the porphyrin moiety of heme was sufficient to mediate suppression of STAT1-dependent responses in human and mouse macrophages and promoted liver dissemination of K. pneumoniae in vivo. Thus, cellular heme metabolism dysfunction negatively regulates the STAT1 pathway with implications in severe infection.

Purpose Nitrate (NO3-), through its conversion to nitrite (NO2-) and nitric oxide, has been shown to increase exercise tolerance in healthy younger adults and older diseased patients. Nitrate’s effect in well-trained middle to older-aged adults has not been studied. Therefore, the purpose of this investigation was to examine the effects of a NO3- rich beverage on submaximal constant work rate exercise time in well-trained middle to older-aged adults. Methods This was a randomized controlled cross-over trial with 15 well-trained middle to older-aged adults, 41–64 year-old, who received one of two treatments (NO3- rich beverage then placebo or placebo then NO3- rich beverage), after which an exercise test at 75 percent of the subject’s maximal work rate was completed. Results The NO3- rich beverage increased plasma NO3- and NO2- levels by 260 μM and 0.47 μM, respectively (p

Background Storage temperature is a critical factor for maintaining red-blood cell (RBC) viability, especially during prolonged cold storage. The target range of 1 to 6°C was established decades ago and may no longer be optimal for current blood-banking practices. Study design and methods Human and canine RBCs were collected under standard conditions and stored in precision-controlled refrigerators at 2°C, 4°C, or 6°C. Results During 42-day storage, human and canine RBCs showed progressive increases in supernatant non-transferrin-bound iron, cell-free hemoglobin, base deficit, and lactate levels that were overall greater at 6°C and 4°C than at 2°C. Animals transfused with 7-day-old RBCs had similar plasma cell-free hemoglobin and non-transferrin-bound iron levels at 1 to 72 hours for all three temperature conditions by chromium-51 recovery analysis. However, animals transfused with 35-day-old RBCs stored at higher temperatures developed plasma elevations in non-transferrin-bound iron and cell-free hemoglobin at 24 and 72 hours. Despite apparent impaired 35-day storage at 4°C and 6°C compared to 2°C, posttransfusion chromium-51 recovery at 24 hours was superior at higher temperatures. This finding was confounded by a preparation artifact related to an interaction between temperature and storage duration that leads to removal of fragile cells with repeated washing of the radiolabeled RBC test sample and renders the test sample unrepresentative of the stored unit. Conclusions RBCs stored at the lower bounds of the temperature range are less metabolically active and produce less anaerobic acidosis and hemolysis, leading to a more suitable transfusion product. The higher refrigeration temperatures are not optimal during extended RBC storage and may confound chromium viability studies.

Inorganic nitrate (NO(3) (−)), nitrite (NO(2) (−)) and NO are nitrogenous species with a diverse and interconnected chemical biology. The formation of NO from nitrate and nitrite via a reductive ‘nitrate–nitrite–NO’ pathway and resulting in vasodilation is now an established complementary route to traditional NOS‐derived vasodilation. Nitrate, found in our diet and abundant in mammalian tissues and circulation, is activated via reduction to nitrite predominantly by our commensal oral microbiome. The subsequent in vivo reduction of nitrite, a stable vascular reserve of NO, is facilitated by a number of haem‐containing and molybdenum‐cofactor proteins. NO generation from nitrite is enhanced during physiological and pathological hypoxia and in disease states involving ischaemia–reperfusion injury. As such, modulation of these NO vascular repositories via exogenously supplied nitrite and nitrate has been evaluated as a therapeutic approach in a number of diseases. Ultimately, the chemical biology of nitrate and nitrite is governed by local concentrations, reaction equilibrium constants, and the generation of transient intermediates, with kinetic rate constants modulated at differing physiological pH values and oxygen tensions. LINKED ARTICLES: This article is part of a themed section on Nitric Oxide 20 Years from the 1998 Nobel Prize. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.2/issuetoc

Sickle cell disease is caused by a mutant form of hemoglobin that polymerizes under hypoxic conditions which leads to red blood cell (RBC) distortion, calcium-influx mediated RBC dehydration, increased RBC adhesivity, reduced RBC deformability, increased RBC fragility, and hemolysis. These impairments in RBC structure and function result in multifaceted downstream pathology including inflammation, endothelial cell activation, platelet and leukocyte activation and adhesion, and thrombosis, all of which contribute vascular occlusion and substantial morbidity and mortality. Hemoglobin released upon RBC hemolysis scavenges nitric oxide (NO) and generates reactive oxygen species (ROS) and thereby decreases bioavailability of this important signaling molecule. As the endothelium-derived relaxing factor, NO acts as a vasodilator and also decreases platelet, leukocyte, and endothelial cell activation. Thus, low NO bioavailability contributes to pathology in sickle cell disease and its restoration could serve as an effective treatment. Despite its promise, clinical trials based on restoring NO bioavailability have so far been mainly disappointing. However, particular "NO donating" agents such as nitrite, which unlike some other NO donors can improve sickle RBC properties, may yet prove effective.

The capacity of mature red blood cells (RBCs) to respond to oxidative stress is limited due to lack of a full complement of organelles and as such, when faced with an oxidative environment, they rely on their endogenous antioxidant capacity (including superoxide dismutase, catalase, peroxiredoxin and glutathione) to protect against cellular damage. Low blood glutathione activity has been reported in several red cell disorders leading to increased oxidative stress. Targeting oxidative stress has thus been proposed as a secondary treatment in multiple anemia-causing diseases, such as sickle cell disease (SCD) and malaria, although its overall efficacy remains unclear. As glutathione itself is not permeable through the RBC membrane, treatment with cell-permeable amino acid precursors of glutathione (glutamine, cysteine and/or glycine) is a potential strategy to expand the RBC's antioxidant capacity and alleviate oxidative stress. Indeed, L-glutamine has recently been approved as a therapeutic for SCD, although the mechanistic basis for its effect is not clear. To fill this gap in our understanding, we performed detailed characterization of biophysical phenotype, morphology, and intracellular redox environment of oxidatively stressed RBCs in environments with varying amounts of available precursor amino acids. To assess the impact of exogenous amino acid precursors on the RBC's glutathione antioxidant capacity, we exposed mature RBCs from healthy adults to hydrogen peroxide (H 2O 2) and co-incubated with media that included glutamine, cysteine, and/or glycine. As catalase has the ability to scavenge high levels of exogenously fluxed H 2O 2, we performed these experiments using sodium azide to block catalase activity, enabling us to model oxidatively stressed RBCs. We performed osmotic gradient ektacytometry to quantify RBC deformability and hydration status, and assessed RBC morphology using osmotic-adjusted fixation techniques and scanning electron microscopy. As previously documented, H 2O 2 exposure in sodium azide-treated healthy RBCs was associated with decreased deformability, decreased hydration and increased numbers of echinocytes in a dose-dependent manner. We monitored red cell phenotypic changes following co-incubation with glutamine, cysteine, and/or glycine individually and in combination to test whether these amino acids extended the RBC's antioxidant abilities and contributed to improved function and morphology. We found that supplementation with all three amino acids in combination significantly improved both deformability and hydration of H 2O 2-stressed RBCs, as opposed to treatment with glutamine alone. To directly assess whether the exogenous amino acids were in fact contributing to less intracellular oxidative stress in RBCs, we quantified intracellular reactive oxygen species (ROS) using 2', 7' -dichlorofluorescein diacetate (DCFDA), a cell permeable dye used to measure ROS production. As expected, H 2O 2 exposure alone was associated with elevated intracellular ROS inside RBCs in both a time- and dose-dependent manner. Supplementation with the three amino acid cocktail during H 2O 2 stress resulted in a reduction in the level of intracellular ROS activity. In summary, we documented that exogenous added amino acids reduce oxidative damage in RBCs and we hypothesize that this protection occurs via the glutathione antioxidant pathways. In future studies, we plan to investigate the impact of exogenous amino acids on sickled and irreversibly sickled RBCs (ISCs) in the context of SCD, and on uninfected and infected RBCs in the context of malaria. Disclosures Kim-Shapiro: Beverage Operations LLC: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: co-inventor on a patent related to the use of nitrite under cardiovascular conditions, and a co-author on patents related to treatment of hemolysis.

Sickle cell disease is caused by a mutant form of hemoglobin that polymerizes under hypoxic conditions, increasing rigidity, fragility, calcium influx-mediated dehydration, and adhesivity of red blood cells. Increased red cell fragility results in hemolysis, which reduces nitric oxide (NO) bioavailability, and induces platelet activation and inflammation leading to adhesion of circulating blood cells. Nitric Oxide inhibits adhesion and platelet activation. Nitrite has emerged as an attractive therapeutic agent that targets delivery of NO activity to areas of hypoxia through bioactivation by deoxygenated red blood cell hemoglobin. In this study, we demonstrate anti-platelet activity of nitrite at doses achievable through dietary interventions with comparison to similar doses with other NO donating agents. Unlike other NO donating agents, nitrite activity is shown to be potentiated in the presence of red blood cells in hypoxic conditions. We also show that nitrite reduces calcium associated loss of phospholipid asymmetry that is associated with increased red cell adhesion, and that red cell deformability is also improved. We show that nitrite inhibits red cell adhesion in a microfluidic flow-channel assay after endothelial cell activation. In further investigations, we show that leukocyte and platelet adhesion is blunted in nitrite-fed wild type mice compared to control after either lipopolysaccharide- or hemolysis-induced inflammation. Moreover, we demonstrate that nitrite treatment results in a reduction in adhesion of circulating blood cells and reduced red blood cell hemolysis in humanized transgenic sickle cell mice subjected to local hypoxia. These data suggest that nitrite is an effective anti-platelet and anti-adhesion agent that is activated by red blood cells, with enhanced potency under physiological hypoxia and in venous blood that may be useful therapeutically., Graphical abstract fx1, Highlights • Nitrite is a unique inhibitor of platelet activation bioactivated by RBCs in hypoxia. • Nitrite improves RBC properties following calcium-influx induced damage. • Nitrite blunts hemolysis and inflammation induced adhesion of blood cells. • Nitrite blunts adhesion of circulating blood cells in a sickle cell mouse model. • Nitrite may be harnessed therapeutically in sickle cell disease.

BACKGROUND No studies have been performed comparing intravenous (IV) iron with transfused red blood cells (RBCs) for treating anemia during infection. In a previous report, transfused older RBCs increased free iron release and mortality in infected animals when compared to fresher cells. We hypothesized that treating anemia during infection with transfused fresh RBCs, with minimal free iron release, would prove superior to IV iron therapy. STUDY DESIGN AND METHODS Purpose-bred beagles (n = 42) with experimental Staphylococcus aureus pneumonia rendered anemic were randomized to be transfused RBCs stored for 7 days or one of two IV iron preparations (7 mg/kg), iron sucrose, a widely used preparation, or ferumoxytol, a newer formulation that blunts circulating iron levels. RESULTS Both irons increased the alveolar-arterial oxygen gradient at 24 to 48 hours (p = 0.02-0.001), worsened shock at 16 hours (p = 0.02-0.003, respectively), and reduced survival (transfusion 56%; iron sucrose 8%, p = 0.01; ferumoxytol 9%, p = 0.04). Compared to fresh RBC transfusion, plasma iron measured by non-transferrin-bound iron levels increased with iron sucrose at 7, 10, 13, 16, 24, and 48 hours (p = 0.04 to p

Background During sepsis, higher plasma cell-free hemoglobin (CFH) levels portend worse outcomes. In sepsis models, plasma proteins that bind CFH improve survival. In our canine antibiotic-treated Staphylococcus aureus pneumonia model, with and without red blood cell (RBC) exchange transfusion, commercial human haptoglobin (Hp) concentrates bound and compartmentalized CFH intravascularly, increased CFH clearance, and lowered iron levels, improving shock, lung injury, and survival. We now investigate in our model how very high CFH levels and treatment time affect Hp's beneficial effects. Materials and methods Two separate canine pneumonia sepsis Hp studies were undertaken: one with exchange transfusion of RBCs after prolonged storage to raise CFH to very high levels and another with rapidly lethal sepsis alone to shorten time to treat. All animals received continuous standard intensive care unit supportive care for 96 hours. Results Older RBCs markedly elevated plasma CFH levels and, when combined with Hp therapy, created supraphysiologic CFH-Hp complexes that did not increase CFH or iron clearance or improve lung injury and survival. In a rapidly lethal bacterial challenge model without RBC transfusion, Hp binding did not increase clearance of complexes or iron or show benefits seen previously in the less lethal model. Discussion High-level CFH-Hp complexes may impair clearance mechanisms and eliminate Hp's beneficial effect during sepsis. Rapidly lethal sepsis narrows the therapeutic window for CFH and iron clearance, also decreasing Hp's beneficial effects. In designing clinical trials, dosing and kinetics may be critical factors if Hp infusion is used to treat sepsis.

Nitrite was once thought to be inert in human physiology. However, research over the past few decades has established a link between nitrite and the production of nitric oxide (NO) that is potentiated under hypoxic and acidic conditions. Under this new role nitrite acts as a storage pool for bioavailable NO. The NO so produced is likely to play important roles in decreasing platelet activation, contributing to hypoxic vasodilation and minimizing blood-cell adhesion to endothelial cells. Researchers have proposed multiple mechanisms for nitrite reduction in the blood. However, NO production in blood must somehow overcome rapid scavenging by hemoglobin in order to be effective. Here we review the role of red blood cell hemoglobin in the reduction of nitrite and present recent research into mechanisms that may allow nitric oxide and other reactive nitrogen signaling species to escape the red blood cell.

Background Transfusion of blood at the limits of approved storage time is associated with lower red blood cell (RBC) post-transfusion recovery and hemolysis, which increases plasma cell-free hemoglobin and iron, proposed to induce endothelial dysfunction and impair host defense. There is noted variability among donors in the intrinsic rate of storage changes and RBC post-transfusion recovery, yet genetic determinants that modulate this process are unclear. Methods We explore RBC storage stability and post-transfusion recovery in murine models of allogeneic and xenogeneic transfusion using blood from humanized transgenic sickle cell hemizygous mice (Hbatm1PazHbbtm1TowTg(HBA-HBBs)41Paz/J) and human donors with a common genetic mutation sickle cell trait (HbAS). Findings Human and transgenic HbAS RBCs demonstrate accelerated storage time-dependent hemolysis and reduced post-transfusion recovery in mice. The rapid post-transfusion clearance of stored HbAS RBC is unrelated to macrophage-mediated uptake or intravascular hemolysis, but by enhanced sequestration in the spleen, kidney and liver. HbAS RBCs are intrinsically different from HbAA RBCs, with reduced membrane deformability as cells age in cold storage, leading to accelerated clearance of transfused HbAS RBCs by entrapment in organ microcirculation. Interpretation The common genetic variant HbAS enhances RBC storage dysfunction and raises provocative questions about the use of HbAS RBCs at the limits of approved storage., Highlights • Sickle cell trait (HbAS) RBC exhibit increased resistance to osmotic shock compared to normal (HbAA) RBCs. • HbAS RBC show accelerated storage-related aging and post-transfusion clearance after cold storage compared to HbAA RBC. • Reduced post-transfusion survival of stored HbAS RBCs is not due to intravascular hemolysis but due to tissue sequestration. In allogeneic transfusions, red blood cells (RBCs) are collected and stored for up to 42 days. Historically, donor RBC genetic background is only considered in the context of major Rh and ABO blood groups. This study shows that donor-specific genetic factors such as sickle cell trait, the benign heterozygote state of sickle cell disease, accelerate storage-related hemolysis and reduces RBC post-transfusion survival in mice. Impaired post-transfusion recovery is due to enhanced sequestration in organ microcirculation. Further studies are warranted to determine an appropriate earlier outdate for HbAS RBC units, particularly in malaria-endemic regions where sickle cell trait prevalence is high.