Nitrite and nitrate chemical biology and signalling DeMartino, Anthony W; Kim‐Shapiro, Daniel B.; Patel, Rakesh P ...
British journal of pharmacology,
January 2019, Letnik:
176, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Inorganic nitrate (NO3−), nitrite (NO2−) 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
As stored blood ages intraerythrocytic energy sources are depleted resulting in reduced structural integrity of the membrane. Thus, stored red blood cells (RBCs) become less deformable and more ...fragile as they age. This fragility leads to release of cell‐free hemoglobin (Hb) and formation of microparticles, submicron Hb‐containing vesicles. Upon transfusion, it is likely that additional hemolysis and microparticle formation occurs due to breakdown of fragile RBCs. Release of cell‐free Hb and microparticles leads to increased consumption of nitric oxide (NO), an important signaling molecule that modulates blood flow, and may promote inflammation. Stored blood may also be deficient in recently discovered blood NO synthase activity. We hypothesize that these factors play a potential role in the blood storage lesion.
The rate that hemoglobin reacts with nitric oxide (NO) is limited by how fast NO can diffuse into the heme pocket. The reaction is as fast as any ligand/protein reaction can be and the result, when ...hemoglobin is in its oxygenated form, is formation of nitrate in what is known as the dioxygenation reaction. As nitrate, at the concentrations made through the dioxygenation reaction, is biologically inert, the only role hemoglobin was once thought to play in NO signaling was to inhibit it. However, there are now several mechanisms that have been discovered by which hemoglobin may preserve, control, and even create NO activity. These mechanisms involve compartmentalization of reacting species and conversion of NO from or into other species such as nitrosothiols or nitrite which could transport NO activity. Despite the tremendous amount of work devoted to this field, major questions concerning precise mechanisms of NO activity preservation as well as if and how Hb creates NO activity remain unanswered.
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..• Hemoglobin reacts rapidly with NO eliminating the bioactivity of NO.• Compartmentalization of hemoglobin inside the red cell preserves NO bioactivity.• Oxidation of hemoglobin to methemoglobin preserves NO bioactivity.• Nitrite and hemoglobin can react to form NO and other bioactive NO species.
Chromosomal rearrangements of the gene encoding ROS1 proto-oncogene receptor tyrosine kinase (ROS1) define a distinct molecular subgroup of non-small-cell lung cancers (NSCLCs) that may be ...susceptible to therapeutic ROS1 kinase inhibition. Crizotinib is a small-molecule tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK), ROS1, and another proto-oncogene receptor tyrosine kinase, MET.
We enrolled 50 patients with advanced NSCLC who tested positive for ROS1 rearrangement in an expansion cohort of the phase 1 study of crizotinib. Patients were treated with crizotinib at the standard oral dose of 250 mg twice daily and assessed for safety, pharmacokinetics, and response to therapy. ROS1 fusion partners were identified with the use of next-generation sequencing or reverse-transcriptase-polymerase-chain-reaction assays.
The objective response rate was 72% (95% confidence interval CI, 58 to 84), with 3 complete responses and 33 partial responses. The median duration of response was 17.6 months (95% CI, 14.5 to not reached). Median progression-free survival was 19.2 months (95% CI, 14.4 to not reached), with 25 patients (50%) still in follow-up for progression. Among 30 tumors that were tested, we identified 7 ROS1 fusion partners: 5 known and 2 novel partner genes. No correlation was observed between the type of ROS1 rearrangement and the clinical response to crizotinib. The safety profile of crizotinib was similar to that seen in patients with ALK-rearranged NSCLC.
In this study, crizotinib showed marked antitumor activity in patients with advanced ROS1-rearranged NSCLC. ROS1 rearrangement defines a second molecular subgroup of NSCLC for which crizotinib is highly active. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).
Blood transfusion represents the first and most prescribed cell-based therapy; however, clinical safety and efficacy trials are lacking. Clinical cohort studies have suggested that massive ...transfusion and/or transfusion of aged stored blood may contribute to multiorgan dysfunction in susceptible patients. In this issue of the JCI, Baek and colleagues report that aged stored blood hemolyzes after massive transfusion in a guinea pig model. Hemolysis led to vascular and kidney injury that was mediated by cell-free plasma hemoglobin and prevented by coinfusion of the specific hemoglobin scavenger protein, haptoglobin. These studies support an expanding body of research indicating that intravascular hemolysis is a pathological mechanism in several human diseases, including multiorgan dysfunction after either massive red blood cell transfusion or hemoglobin-based blood substitute therapy, the hemoglobinopathies, malaria, and other acquired and genetic hemolytic conditions.
Erythrocytes regulate vascular function through the modulation of oxygen delivery and the scavenging and generation of nitric oxide (NO). First, hemoglobin inside the red blood cell binds oxygen in ...the lungs and delivers it to tissues throughout the body in an allosterically regulated process, modulated by oxygen, carbon dioxide and proton concentrations. The vasculature responds to low oxygen tensions through vasodilation, further recruiting blood flow and oxygen carrying erythrocytes. Research has shown multiple mechanisms are at play in this classical hypoxic vasodilatory response, with a potential role of red cell derived vasodilatory molecules, such as nitrite derived nitric oxide and red blood cell ATP, considered in the last 20 years. According to these hypotheses, red blood cells release vasodilatory molecules under low oxygen pressures. Candidate molecules released by erythrocytes and responsible for hypoxic vasodilation are nitric oxide, adenosine triphosphate and S-nitrosothiols. Our research group has characterized the biochemistry and physiological effects of the electron and proton transfer reactions from hemoglobin and other ferrous heme globins with nitrite to form NO. In addition to NO generation from nitrite during deoxygenation, hemoglobin has a high affinity for NO. Scavenging of NO by hemoglobin can cause vasoconstriction, which is greatly enhanced by cell free hemoglobin outside of the red cell. Therefore, compartmentalization of hemoglobin inside red blood cells and localization of red blood cells in the blood stream are important for healthy vascular function. Conditions where erythrocyte lysis leads to cell free hemoglobin or where erythrocytes adhere to the endothelium can result in hypertension and vaso constriction. These studies support a model where hemoglobin serves as an oxido-reductase, inhibiting NO and promoting higher vessel tone when oxygenated and reducing nitrite to form NO and vasodilate when deoxygenated.
Background
Ex vivo labeling with 51chromium represents the standard method to determine red blood cell (RBC) survival after transfusion. Limitations and safety concerns spurred the development of ...alternative methods, including biotinylated red blood cells (BioRBC).
Study Design and Methods
Autologous 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).
Results
Data were consistent with biphasic exponential clearance of cells of uniform age. Our best estimate of BioRBC clearance (half‐life T1/2) was 49.7 ± 1.2 days initially, followed by more rapid clearance 82 days after transfusion (T1/2 = 15.6 ± 0.6 days). As BioRBC aged in vivo, molecules of equivalent soluble fluorochrome declined with a T1/2 of 122 ± 9 days, suggesting gradual biotin cleavage. There were no significant differences between the clearance of fresh and aged BioRBC.
Conclusion
Similar 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.
Background
Donor genetic variation is associated with red blood cell (RBC) storage integrity and post‐transfusion recovery. Our previous large‐scale genome‐wide association study demonstrated that ...the African G6PD deficient A‐ variant (rs1050828, Val68Met) is associated with higher oxidative hemolysis after cold storage. Despite a high prevalence of X‐linked G6PD mutation in African American population (>10%), blood donors are not routinely screened for G6PD status and its importance in transfusion medicine is relatively understudied.
Study Design and Methods
To further evaluate the functional effects of the G6PD A‐ mutation, we created a novel mouse model carrying this genetic variant using CRISPR‐Cas9. We hypothesize that this humanized G6PD A‐ variant is associated with reduced G6PD activity with a consequent effect on RBC hemolytic propensity and post‐transfusion recovery.
Results
G6PD A‐ RBCs had reduced G6PD protein with ~5% residual enzymatic activity. Significantly increased in vitro hemolysis induced by oxidative stressors was observed in fresh and stored G6PD A‐ RBCs, along with a lower GSH:GSSG ratio. However, no differences were observed in storage hemolysis, osmotic fragility, mechanical fragility, reticulocytes, and post‐transfusion recovery. Interestingly, a 14% reduction of 24‐h survival following irradiation was observed in G6PD A‐ RBCs compared to WT RBCs. Metabolomic assessment of stored G6PD A‐ RBCs revealed an impaired pentose phosphate pathway (PPP) with increased glycolytic flux, decreasing cellular antioxidant capacity.
Discussion
This novel mouse model of the common G6PD A‐ variant has impaired antioxidant capacity like humans and low G6PD activity may reduce survival of transfused RBCs when irradiation is performed.
Hemoglobin and myoglobin are among the most extensively studied proteins, and nitrite is one of the most studied small molecules. Recently, multiple physiologic studies have surprisingly revealed ...that nitrite represents a biologic reservoir of NO that can regulate hypoxic vasodilation, cellular respiration, and signaling. These studies suggest a vital role for deoxyhemoglobin- and deoxymyoglobin-dependent nitrite reduction. Biophysical and chemical analysis of the nitrite-deoxyhemoglobin reaction has revealed unexpected chemistries between nitrite and deoxyhemoglobin that may contribute to and facilitate hypoxic NO generation and signaling. The first is that hemoglobin is an allosterically regulated nitrite reductase, such that oxygen binding increases the rate of nitrite conversion to NO, a process termed R-state catalysis. The second chemical property is oxidative denitrosylation, a process by which the NO formed in the deoxyhemoglobin-nitrite reaction that binds to other deoxyhemes can be released due to heme oxidation, releasing free NO. Third, the reaction undergoes a nitrite reductase/anhydrase redox cycle that catalyzes the anaerobic conversion of 2 molecules of nitrite into dinitrogen trioxide (N2O3), an uncharged molecule that may be exported from the erythrocyte. We will review these reactions in the biologic framework of hypoxic signaling in blood and the heart.
Since 2016, over half of the states in the United States have passed mandatory limits on opioid prescriptions, with limited evidence of effectiveness. In this study, we evaluated postoperative opioid ...prescriptions following orthopaedic surgery before and after the implementation of one of the earliest such laws.
Following the implementation of state legislation limiting opioid prescriptions for opioid-naïve patients, 2 patient cohorts (pre-law and post-law) were compared. Both opioid-tolerant and opioid-naïve patients undergoing 6 common orthopaedic procedures (total knee arthroplasty, rotator cuff repair, anterior cruciate ligament reconstruction, open reduction and internal fixation for a distal radial fracture, open reduction and internal fixation for an ankle fracture, and lumbar discectomy) met inclusion criteria. Patients undergoing >1 primary procedure in the same operative session were excluded. All benzodiazepine and opioid prescriptions from 30 days before to 90 days after the surgical procedure were recorded. Logistic regression was performed to determine risk factors for prolonged postoperative opioid use.
In this study, 836 pre-law patients were compared with 940 post-law patients. The 2 groups were similar with regard to demographic variables, baseline opioid tolerance, and recent benzodiazepine use (all p > 0.05). Post-law, for all patients, there were decreases in the initial prescription pill quantity (49.65 pills pre-law and 22.08 pills post-law; p < 0.001) and the total morphine milligram equivalents (MMEs) (417.67 MMEs pre-law and 173.86 MMEs post-law; p < 0.001), regardless of patient preoperative opioid exposure (all p < 0.001). Additionally, there were decreases in the mean cumulative 30-day MMEs (790.01 MMEs pre-law and 524.61 MMEs post-law; p < 0.001) and the 30 to 90-day MMEs (243.51 MMEs pre-law and 208.54 MMEs post-law; p = 0.008). Despite being specifically exempted from the legislation, opioid-tolerant patients likewise experienced a significant decrease in cumulative 30-day MMEs (1,304.08 MMEs pre-law and 1,015.19 MMEs post-law; p = 0.0016). Opioid-tolerant patients required more postoperative opioids at all time points and had an increased likelihood of prolonged opioid use compared with those who were opioid-naïve preoperatively (odds ratio, 8.73 95% confidence interval, 6.21 to 12.29).
A clinically important and significant reduction in opioid utilization after orthopaedic surgery was observed following the implementation of statewide mandatory opioid prescription limits.
After implementation of mandatory opioid prescription regulations, a clinically important and significant decline in the volume of opioids dispensed in the short term and intermediate term following orthopaedic surgery was observed. Furthermore, important clinical predictors of prolonged postoperative opioid use, including preoperative opioid use and preoperative benzodiazepine use, were identified. These findings have important implications for public health, as well as the potential to influence policymakers and to change practice among orthopaedic surgeons.
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