Plasma Nitric Oxide Consumption Research Articles

Plasma Nitric Oxide Consumption Is Elevated and Associated With Adverse Outcomes in Critically Ill Patients.

Impaired nitric oxide (NO) bioavailability may contribute to microvascular dysfunction in sepsis. Excessive plasma NO consumption has been attributed to scavenging by circulating cell-free hemoglobin. This may be a mechanism for NO deficiency in sepsis and critical illness. We hypothesized that plasma NO consumption is high in critically ill patients, particularly those with sepsis, acute respiratory distress syndrome (ARDS), shock, and in hospital nonsurvivors. We further hypothesized that plasma NO consumption is correlated with plasma cell-free hemoglobin concentration. Retrospective cohort study. Adult ICUs of an academic medical center. Three hundred sixty-two critically ill patients and 46 healthy control subjects. None. Plasma NO consumption was measured using reductive chemiluminescence and cell-free hemoglobin was measured with a colorimetric assay. Mean (95% CI) plasma NO consumption (µM) was higher in critically ill patients versus healthy control subjects (3.9 [3.7-4.1] vs 2.1 [1.8-2.5]), septic versus nonseptic patients (4.1 [3.8-4.3] vs 3.6 [3.3-3.8]), ARDS versus non-ARDS patients (4.4 [4.0-4.9] vs 3.7 [3.6-3.9]), shock vs nonshock patients (4.4 [4.0-4.8] vs 3.6 [3.4-3.8]), and hospital nonsurvivors versus survivors (5.3 [4.4-6.4] vs 3.7 [3.6-3.9]). These relationships remained significant in multivariable analyses. Plasma cell-free hemoglobin was weakly correlated with plasma NO consumption. Plasma NO consumption is elevated in critically ill patients and independently associated with sepsis, ARDS, shock, and hospital death. These data suggest that excessive intravascular NO scavenging characterizes sepsis and adverse outcomes of critical illness.

Pulmonary and Systemic Vascular Resistances After Cardiopulmonary Bypass: Role of Hemolysis

Prolonged cardiopulmonary bypass (CPB) is associated with hemolysis, resulting in increased plasma oxyhemoglobin and vascular nitric oxide depletion. The authors hypothesized that hemolysis associated with CPB would reduce nitric oxide bioavailability, resulting in high pulmonary and systemic vascular resistances that after CPB would normalize gradually over time, due to clearance of plasma oxyhemoglobin. The authors also investigated whether prolonged CPB (≥140 min) produced increased levels of hemolysis and greater pulmonary and systemic vasoconstriction. Prospective cohort study. Single-center university hospital. The study comprised 50 patients undergoing elective cardiac surgery requiring CPB. Plasma hemoglobin and plasma nitric oxide consumption were measured before surgery and after CPB. Pulmonary and systemic hemodynamics were measured after CPB. The effects of short (<140 min) and prolonged (≥140 min) CPB on these parameters were considered. Pulmonary and systemic vascular resistances and plasma hemoglobin and nitric oxide consumption were highest at 15 minutes after CPB and then decreased over time. Pulmonary and systemic vascular resistances and plasma hemoglobin and plasma nitric oxide consumption were higher in patients requiring prolonged CPB. The reduction in plasma nitric oxide consumption from 15 minutes to 4 hours after CPB was correlated independently with the reductions in pulmonary and systemic vascular resistances. Prolonged CPB was associated with increased plasma hemoglobin and plasma nitric oxide consumption and pulmonary and systemic vascular resistances. The reduction in plasma nitric oxide consumption at 4 hours after CPB was an independent predictor of the concomitant reductions in pulmonary and systemic vascular resistances.

Hemolysis during cardiac surgery is associated with increased intravascular nitric oxide consumption and perioperative kidney and intestinal tissue damage

Introduction: Acute kidney injury (AKI) and intestinal injury negatively impact patient outcome after cardiac surgery. Enhanced nitric oxide (NO) consumption due to intraoperative intravascular hemolysis, may play an important role in this setting. This study investigated the impact of hemolysis on plasma NO consumption, AKI, and intestinal tissue damage, after cardiac surgery.Methods: Hemolysis (by plasma extracellular (free) hemoglobin; fHb), plasma NO-consumption, plasma fHb-binding capacity by haptoglobin (Hp), renal tubular injury (using urinary N-Acetyl-β-D-glucosaminidase; NAG), intestinal mucosal injury (through plasma intestinal fatty acid binding protein; IFABP), and AKI were studied in patients undergoing off-pump cardiac surgery (OPCAB, N = 7), on-pump coronary artery bypass grafting (CABG, N = 30), or combined CABG and valve surgery (CABG+Valve, N = 30).Results: FHb plasma levels and NO-consumption significantly increased, while plasma Hp concentrations significantly decreased in CABG and CABG+Valve patients (p < 0.0001) during surgery. The extent of hemolysis and NO-consumption correlated significantly (r2 = 0.75, p < 0.0001). Also, NAG and IFABP increased in both groups (p < 0.0001, and p < 0.001, respectively), and both were significantly associated with hemolysis (Rs = 0.70, p < 0.0001, and Rs = 0.26, p = 0.04, respectively) and NO-consumption (Rs = 0.55, p = 0.002, and Rs = 0.41, p = 0.03, respectively), also after multivariable logistic regression analysis. OPCAB patients did not show increased fHb, NO-consumption, NAG, or IFABP levels. Patients suffering from AKI (N = 9, 13.4%) displayed significantly higher fHb and NAG levels already during surgery compared to non-AKI patients.Conclusions: Hemolysis appears to be an important contributor to postoperative kidney injury and intestinal mucosal damage, potentially by limiting NO-bioavailability. This observation offers a novel diagnostic and therapeutic target to improve patient outcome after cardiothoracic surgery.

Influence Of Inhaled Nitric Oxide On Stored Red Blood Cell autotransfusion In Overweight Volunteers With Endothelial Dysfunction: Preliminary Results

IntroductionTransfusion of stored packed erythrocytes is associated with excessive intra and extravascular hemolysis. High plasma cell-free hemoglobin levels rapidly scavenge endogenous nitric oxide (NO) leading in many species to systemic and pulmonary vasoconstriction, and a pro-inflammatory, pro-thrombotic state. MethodsWe conducted a crossover randomized interventional study of autologous stored blood transfusion. We enrolled fourteen overweight adults (BMI 28 to 40 Kg/m2) who also had evidence of impaired endothelial function.Endothelial dysfunction was determined by a reduced Reactive Hyperemia Index following 5 minutes of forearm tourniquet ischemia (RHI<1.8).Over 8 months each volunteer returned to the blood bank on 3 occasions to donate a unit of blood. In a randomized scheme,autologous blood was leukoreduced and stored in AS-3, and re-infused after a) 3-days of storage (3-d), b) 40-days of storage (40-d), or c) 40-days of storage and breathing 80 parts per million (ppm) NO (40-d+NO).Venous blood was sampled before and at 10min, 1h, 2h, 4h and 24h after autotransfusion.Plasma hemoglobin measurement Plasma hemoglobin was measured with a QuantiChrom Hemoglobin Assay Kit (BioAssaySystems, Hayward, CA). These samples were collected using 16 G catheters avoiding negative aspirating pressures to minimize ex vivo hemolysis (1).Plasma NO consumption The ability of plasma hemoglobin to scavengeNO was measured with an NO consumption assay with a NO chemiluminescenceanalyzer (Sievers, Boulder, CO) (2) ResultsEleven volunteers completed the entire study. Volunteer characteristics at baseline were (mean ± SD): age: 41± 13 years; sex: 9 males, 5 females; BMI 33.2 ± 4.9 Kg/m2; reactive hyperemia index 1.59±0.18, and plasma levels of C-Reactive Protein 4.75 ±3.44 md/L.After autotransfusion the plasma hemoglobin and serum iron concentrations were increased from 1 to 4 hours after transfusing 40-days stored blood. No increase was detected after transfusing 3-days stored blood (p<0.01 value differs from b and c) (Figures 1a and 2). [Display omitted] [Display omitted] The plasma NO consumption assay showed an increased level in the 40-d group (p<0.05 value differs from both b and c) (Figure 1b). Plasma concentrations of potassium, lactate dehydrogenase, haptoglobin, cytokines, and systemic blood pressure did not differ between a, b and c and all remained within the normal range. ConclusionsTransfusion of autologous packed erythrocytes stored for 40 days in AS3 is associated with increased hemolysis and increased plasma NO consumption,the latter is prevented by breathing 80 ppm NO.

Decreased nitrite levels in erythrocytes of children with β-thalassemia/hemoglobin E

Nitrite anion is bioactive nitric oxide (NO) species circulating in blood, and represents the NO bioavailability and endothelial function. In this study, we aimed to investigate the nitrite levels and the correlation with hemolysis and severity in β-thalassemia/hemoglobin E (β-thal/HbE). 38 Children (12.0±1.9years of age) with a diagnosis of mild, moderate and severe β-thalassemia were enrolled in the study. The blood nitrite levels and potential plasma NO consumption were measured by the chemiluminescence method. The nitrite levels in whole blood and erythrocytes of the severe thalassemia subjects were lower than those of the control subjects. At day 7 after transfusion of packed erythrocytes, the nitrite levels in erythrocytes increased. The plasma hemoglobin and NO consumption increased in the severe thalassemia subjects. The nitrite levels in erythrocytes inversely correlated with plasma hemoglobin, lactate dehydrogenase activity, potential NO consumption, and lipid peroxidation. Our studies demonstrate the decreased NO bioavailability in thalassemia, which could result from endothelial dysfunction, the increased potential NO consumption in plasma by cell-free hemoglobin and oxidative stress.

Blood transfusions increase circulating plasma free hemoglobin levels and plasma nitric oxide consumption: a prospective observational pilot study

IntroductionThe increasing number of reports on the relation between transfusion of stored red blood cells (RBCs) and adverse patient outcome has sparked an intense debate on the benefits and risks of blood transfusions. Meanwhile, the pathophysiological mechanisms underlying this postulated relation remain unclear. The development of hemolysis during storage might contribute to this mechanism by release of free hemoglobin (fHb), a potent nitric oxide (NO) scavenger, which may impair vasodilation and microcirculatory perfusion after transfusion. The objective of this prospective observational pilot study was to establish whether RBC transfusion results in increased circulating fHb levels and plasma NO consumption. In addition, the relation between increased fHb values and circulating haptoglobin, its natural scavenger, was studied.MethodsThirty patients electively received 1 stored packed RBC unit (n = 8) or 2 stored packed RBC units (n = 22). Blood samples were drawn to analyze plasma levels of fHb, haptoglobin, and NO consumption prior to transfusion, and 15, 30, 60 and 120 minutes and 24 hours after transfusion. Differences were compared using Pearson's chi-square test or Fisher's exact test for dichotomous variables, or an independent-sample t test or Mann-Whitney U test for continuous data. Continuous, multiple-timepoint data were analyzed using repeated one-way analysis of variance or the Kruskall-Wallis test. Correlations were analyzed using Spearman or Pearson correlation.ResultsStorage duration correlated significantly with fHb concentrations and NO consumption within the storage medium (r = 0.51, P < 0.001 and r = 0.62, P = 0.002). fHb also significantly correlated with NO consumption directly (r = 0.61, P = 0.002). Transfusion of 2 RBC units significantly increased circulating fHb and NO consumption in the recipient (P < 0.001 and P < 0.05, respectively), in contrast to transfusion of 1 stored RBC unit. Storage duration of the blood products did not correlate with changes in fHb and NO consumption in the recipient. In contrast, pre-transfusion recipient plasma haptoglobin levels inversely influenced post-transfusion fHb concentrations.ConclusionThese data suggest that RBC transfusion can significantly increase post-transfusion plasma fHb levels and plasma NO consumption in the recipient. This finding may contribute to the potential pathophysiological mechanism underlying the much-discussed adverse relation between blood transfusions and patient outcome. This observation may be of particular importance for patients with substantial transfusion requirements.

Increased Pulmonary Pressures and Myocardial Wall Stress in Children with Severe Malaria

Chronic intravascular hemolysis leads to nitric oxide (NO) depletion and pulmonary hypertension in sickle cell disease. To test whether this pathophysiology occurs in malaria, we examined in Mali 53 children who were admitted to the hospital with severe malaria (excluding cerebral malaria) and 31 age-matched controls. Severity of hemolysis was assessed from plasma levels of free hemoglobin and arginase-1. NO metabolism was assessed by whole-blood nitrite levels and plasma NO consumption. Effects on the cardiovascular system and endothelial function were assessed by using echocardiography to measure peak tricuspid regurgitant jet velocity and by evaluating plasma levels of N-terminal prohormone brain natriuretic peptide (NT-proBNP) and soluble vascular cell adhesion molecule-1. Children with severe malaria had higher plasma levels of hemoglobin and arginase-1, reduced whole-blood levels of nitrite, and increased NO consumption relative to controls. They also had increased pulmonary arterial pressures (P< .05) with elevated levels of NT-proBNP and soluble vascular cell adhesion molecule-1 (P< .001). Children with severe malaria have increased pulmonary pressures and myocardial wall stress. These complications are consistent with NO depletion from intravascular hemolysis, and they indicate that the pathophysiologic cascade from intravascular hemolysis to NO depletion and its cardiopulmonary effects is activated in children with severe malaria.

Increased circulating cell-free hemoglobin levels reduce nitric oxide bioavailability in preeclampsia

Contrasting with increased nitric oxide (NO) formation during healthy pregnancy, reduced NO bioavailability plays a role in preeclampsia. However, no study has examined whether increased NO consumption by enhanced circulating levels of cell-free hemoglobin plays a role in preeclampsia. We studied 82 pregnant women (38 healthy pregnant and 44 with preeclampsia). To assess NO bioavailability, we measured plasma and whole blood nitrite concentrations using an ozone-based chemiluminescence assay. Plasma ceruloplasmin concentrations and plasma NO consumption (pNOc) were assessed and plasma hemoglobin (pHb) concentrations were measured with a commercial immunoassay. We found lower whole blood and plasma nitrite concentrations in preeclamptic patients (-48 and -39%, respectively; both P < 0.05) compared with healthy pregnant women. Plasma samples from preeclamptic women consumed 63% more NO ( P = 0.003) and had 53% higher pHb and 10% higher ceruloplasmin levels than those found in healthy pregnant women ( P < 0.01). We found significant positive correlations between pHb and pNOc ( r = 0.61; P < 0.0001), negative correlations between pNOc and whole blood or plasma nitrite concentrations ( P = 0.02; r = -0.32 and P = 0.01; r = -0.34, respectively), and negative correlations between pHb and whole blood or plasma nitrite concentrations ( P = 0.03; r = -0.36 and P = 0.01; r = -0.38, respectively). These findings suggest that increased pHb levels lead to increased NO consumption and lower NO bioavailability in preeclamptic compared with healthy pregnant women.

Mixed haematopoietic chimerism for sickle cell disease prevents intravascular haemolysis

Mixed haematopoietic chimerism for sickle cell disease prevents intravascular haemolysis

Mixed Chimerism Following Nonmyeloablative Stem Cell Transplantation for Sickle Cell Disease Prevents Intravascular Hemolysis and Restores Endothelial Function.

Recent studies have highlighted the impact of chronic intravascular hemolysis on endothelial function and end-organ injury, such as pulmonary hypertension, in patients with sickle cell disease (SCD). Preservation of organ function is a critical criterion of the success of therapy. Nonmyeloablative allogeneic stem cell transplantation (NMA-SCT) is relatively non-toxic and frequently results in mixed hematopoietic chimerism. However, it is not known if mixed chimerism reduces end-organ toxicity. To assess the effects of mixed chimerism on reduction in hemolysis and endothelial function, sequential multiple parameters of intravascular hemolysis were measured in 3 SCD patients who developed mixed chimerism following NMA-SCT. All 3 reported significant improvement in SCD-related symptoms after transplant, despite mixed chimerism. We analyzed serum biomarkers, recently identified to be associated with pulmonary hypertension and endothelial dysfunction, and compared them to chimerism as measured by both routine genomic DNA analysis and by a erythroid lineage specific assay. These include markers of increased hemolytic rate (plasma hemoglobin (Hb), LDH, reticulocytosis), endothelial function (sVCAM-1), and decreased nitric oxide (NO) bioavailability (plasma NO consumption). Analysis of peripheral blood genomic DNA demonstrated 25–50% total donor cell engraftment from d41 to d180 in these patients. During this period of partial donor engraftment, all 3 patients demonstrated improvements in extent of intravascular hemolysis: Median pre to post NMA-SCT levels of LDH decreased 300 U/liter L (range 255–621 U/liter) to 162 U/L (range 122–199 U/liter), and of cell-free Hb concentration decreased from 11.8 uM (range 5.5–15.54) to 3.97 uM (range 1.61–7.75). Similarly, sVCAM-1 levels and NO consumption both decreased from median pre- to post NMA-SCT levels of 712.3 ng/ml (range 654.9–1067) to 413.9 ng/ml (193.9–608.2), and of 3.34 uM (1.91–7.63) to 1.32 uM (0.62–3.92), respectively. After d180, Pt 1 experienced graft rejection, and all hemolysis parameters again approached pre-transplant levels, demonstrating that reduction in intravascular hemolysis was directly attributable to the presence of circulating donor erythrocytes. Studies of RBC chimerism more accurately assessed the functional impact of donor hematopoiesis. RBC chimerism was serially measured by quantifying donor-derived beta-globin RNA in PBMC. Surprisingly, this revealed a ~ 2-fold higher level of donor erythroid precursor expression (70–100%) compared to the level of total donor cell engraftment, attributable to the improved survival of engrafted cells. This near-to full replacement of peripheral erythrocytes with donor derived RBCs accounts for the improvements in hemolysis. Our data reveal that levels of donor engraftment as low as 25% can ameliorate the degree of hemolysis and can potentially protect against ongoing chronic end-organ damage. These improvements have not been previously achievable with any other treatment for SCD, including hydroxyurea. Analysis of additional transplanted SCD patients is ongoing.

Hemolysis-Endothelial Dysfunction Syndrome: A Novel Paradigm of Disease Mechanism.

Background: Normal vasomotor tone is maintained by the production of nitric oxide (NO) from endothelial-derived nitric oxide synthase in excess of consumption of NO within the vasculature by intra-erythrocytic hemoglobin. NO reacts up to 600-fold less rapidly when hemoglobin is compartmentalized within the erythrocyte rather than in a cell free form (i.e. cell-free plasma hemoglobin). This implies that during intravascular hemolysis, NO consumption by hemoglobin would be greatly increased. We have proposed that NO consumption and loss of normal vasomotor tone contributes to the pathogenesis of sickle cell disease. However, the increased reactivity of cell-free plasma hemoglobin, released during hemolysis, compared to intra-erythrocytic hemoglobin and the physiologic consequences of hemolysis on nitric oxide consumption have not been definitively proven in vivo.Methods: We have developed a canine model of free water induced intravascular hemolysis to test this mechanism and to examine whether inhaled NO can selectively oxidize the cell free plasma hemoglobin and thus attenuate any associated physiologic changes. Paired experiments were performed in each animal which included a 6-hour control infusion of 5% Dextrose (D5W) followed one week later by a 6-hour infusion of either D5W or free water with or without 80 ppm inhaled NO (full factorial design). Based on pilot experiments, infusions of free water or D5W (control) were administered at a rate of 16 ml/kg/hr. At the end of each experiment, an escalating sodium nitroprusside infusion, a direct NO donor (1 to 27 mcg/kg/min), was administered to evaluate the vascular responsiveness to NO in the presence and absence of hemolysis. In this model, total intravascular hemoglobin concentration remains constant, whereas the ratio of plasma to intracellular hemoglobin concentration increases with hemolysis.Results: In comparison to D5W, free water infusion caused linear increases in plasma hemoglobin (25–50 mM/h) which remained ferrous (FeII) and correlated with increased plasma nitric oxide consumption (R = 0.95; p<0.001). This free water induced intravascular hemolysis caused significant increases in mean systemic arterial pressure (MAP; p=0.0003) and systemic vascular resistance index (SVRI; p=0.0003) and a significant decrease in cardiac index (CI; p=0.02) compared to an equivalent infusion of D5W. In the setting of hemolysis, concurrent inhalation of nitric oxide gas converted the cell free plasma hemoglobin to methemoglobin (50–90% of total plasma hemoglobin), inhibited NO consumption by plasma (methemoglobin does not oxidize NO to nitrate), and normalized hemodynamic parameters. Furthermore, free water induced hemolysis led to blunted hemodynamic effects of sodium nitroprusside on MAP, CI, and SVRI, which were restored with inhaled nitric oxide therapy (p=0.01, p=0.09, and p=0.003 respectively). This further supports the thesis that cell-free plasma hemoglobin consumes NO in the vasculature.Conclusion: These data support the hypothesis that NO scavenging by cell-free plasma hemoglobin disrupts endothelial NO-dependent vasomotor function producing systemic physiologic changes which are attenuated by inhaled nitric oxide therapy. These biochemical and physiological studies support the existence of a hemolysis-endothelial dysfunction syndrome, which may contribute to the vasculopathy of hereditary, acquired and iatrogenic hemolytic states.