Low micromolar intravascular cell-free hemoglobin concentration affects vascular NO bioavailability in sickle cell disease: a computational analysis

In sickle cell disease, the changes in RBC morphology destabilize the red blood cell (RBC) membrane and lead to hemolysis. Several experimental and clinical studies have associated intravascular hemolysis with pulmonary hypertension in sickle cell disease. Cell-free hemoglobin (Hb) from intravascular hemolysis has high affinity for nitrixc oxide (NO) and can affect the NO bioavailability in the sickle cell disease, which may eventually lead to pulmonary hypertension. To study the effects of intravascular hemolysis related cell-free Hb concentrations on NO bioavailability, we developed a two-dimensional mathematical model of NO biotransport in 50-μm arteriole under steady-state sickle cell disease conditions. We analyzed the effects of flow-dependent NO production and axial and radial transport of NO, a recently reported much lower NO-RBC reaction rate constant, and cell-free layer thickness on NO biotransport. Our results show that the presence of cell-free Hb concentrations as low as 0.5 μM results in an approximately three- to sevenfold reduction in the predicted smooth muscle cell NO concentrations compared with those under physiological conditions. In addition, increasing the diffusional resistance for NO in vascular lumen from cell-free layer or reducing NO-RBC reaction rate did not improve the NO bioavailability at the smooth muscle cell layer significantly for cell-free Hb concentrations ≥1 μM. These results suggest that lower NO bioavailability due to low micromolar cell-free Hb can disturb NO homeostasis and cause insufficient bioavailability at the smooth muscle cell layer. Our results supports the hypothesis that hemolysis-associated reduction in NO bioavailability may play a role in the development of pathophysiological complications like pulmonary hypertension in sickle cell disease that are observed in several clinical and experimental studies.

Download Full-text

Arginine Metabolite Profiling in Sickle Cell Disease: Abnormal Levels and Correlations with Pulmonary Hypertension, Desaturation, Hemolysis and Organ Dysfunction.

Blood ◽

10.1182/blood.v108.11.1205.1205 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1205-1205

Author(s):

Gregory J. Kato ◽

Wang Zeneng ◽

James G. Taylor ◽

Roberto F. Machado ◽

William C. Blackwelder ◽

...

Keyword(s):

Nitric Oxide ◽

Pulmonary Hypertension ◽

Sickle Cell Disease ◽

Renal Dysfunction ◽

Sickle Cell ◽

Early Mortality ◽

Systemic Hypertension ◽

Cell Disease ◽

Intravascular Hemolysis ◽

Arginine Transport

Abstract Pulmonary arterial hypertension (PAH) in patients with sickle cell disease (SCD) is linked to intravascular hemolysis, renal dysfunction, systolic hypertension, cholestasis, and early mortality. Although the pathophysiology of PAH in SCD is multifactorial, one important and fundamental factor is impaired nitric oxide bioavailability. Severe intravascular hemolysis releases hemoglobin and arginase into blood plasma, leading to consumption of nitric oxide and its plasma precursor L-arginine, the obligate substrate for the nitric oxide synthases (NOS). In order to explore other potential alterations in the arginine pathway that might affect arginine bioavailability and nitric oxide production, we used high-performance liquid chromatography-tandem mass spectrometry to determine the plasma concentrations for several key metabolites that may affect NOS activity or arginine transport: asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), N-monomethyl-L-arginine (MMA), and N-ω-hydroxy-L-arginine (NOHA). Plasma levels of ADMA, SDMA and MMA are significantly higher in all forms of SCD than in healthy African American control subjects (Table 1). NOHA, the intermediate species in nitric oxide synthesis from L-arginine, is significantly lower in sickle-β-thalassemia (Sβ-thal) patients and homozygous SCD (SS). L-arginine levels are significantly lower in all forms of SCD, as previously reported. PAH as assessed by echocardiography screening was correlated to SDMA (r=0.30, p<0.0001) and NOHA (r=0.23, p=0.002). Similar correlations were observed to NT-proBNP, another marker of PAH. Low oxygen saturations were linked to high levels of all four arginine metabolites. ADMA levels were elevated with severe hemolysis, and unexpectedly lower with renal dysfunction. Levels of SDMA and NOHA were significantly related to renal dysfunction (p<0.01), with an additional link of NOHA to systemic hypertension (p<0.001). In addition, Cox proportional hazard analysis showed a relationship of the arginine/SDMA ratio to early mortality (p<0.001). In summary, levels of the endogenous NOS inhibitor ADMA are highly elevated in SCD and linked to hemolysis, and may contribute to hemolysis-associated endothelial dysfunction. The levels of SDMA, a competitive inhibitor of arginine transport and intracellular bioavailability, are also elevated and linked to PAH, desaturation, renal dysfunction and early mortality risk. The low levels of arginine and NOHA in SCD are consistent with low substrate availability for NOS, and may also limit NO production. The role of arginine metabolites in dysregulation of the arginine-nitric oxide axis and pulmonary hypertension in SCD merits further investigation. Table 1. Arginine Metabolites in Sickle Cell Disease compared to controls. Metabolite Control (n=29) SC (n=34) Sβ-thal (n=11) SS (n=130) Values indicate median values in μM. *p<0.05; **p<0.01; ***p<0.001, Mann-Whitney test compared to controls. ADMA 0.31 0.82*** 0.92* 0.99*** SDMA 0.83 0.92* 1.03** 1.03*** MMA 0.13 0.15* 0.20** 0.18*** NOHA 2.50 2.23 2.15* 1.80** L-Arginine 78.3 51.5*** 41.6*** 45.5***

Download Full-text

Low Erythrocyte Glutamine-to-Glutamate Ratio: A Novel Biomarker of Hemolysis and Pulmonary Hypertension in Sickle Cell Disease.

Blood ◽

10.1182/blood.v110.11.2257.2257 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2257-2257

Author(s):

Claudia R. Morris ◽

Jung Suh ◽

Ward Hagar ◽

Sandra Larkin ◽

D. Anton Bland ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Sickle Cell Disease ◽

Sickle Cell ◽

De Novo ◽

Glutamine Metabolism ◽

Cell Disease ◽

Total Glutathione ◽

Redox Environment ◽

No Bioavailability ◽

Thiol Redox

Abstract The erythrocyte redox environment may contribute to increased hemolysis and decreased nitric oxide (NO) bioavailability in pulmonary hypertension (PH) of sickle cell disease (SCD). Glutathione (GSH) is the principal thiol redox buffer in erythocytes and its depletion has been linked to hemolysis. Glutamine plays an additional anti-oxidant role through preservation of the intracellular nicotinamide adenine dinucleotide (NAD) levels, required for reducing GSSG back to GSH. We hypothesized that altered GSH and glutamine metabolism promotes hemolysis and contributes to PH in SCD. Glutamine, total glutathione (GSH+GSSG) and its precursors (glutamate, cysteine, glycine) were assayed in plasma and erythrocytes of 40 SCD patients and 9 controls. PH is defined by echocardiogram as a tricuspid regurgitant jet velocity (TRV) ≥ 2.5m/s. Total plasma glutathione was lower in SCD vs control patients (2.7 ± 0.3 μM vs. 4.1± 0.8 μM, p<0.05). Similarly, total erythrocyte glutathione levels were decreased in SCD vs. control patients (310 ± 26 μM vs. 683 ± 110 μM, p<0.0001). A trend towards higher GSH precursor levels identified in plasma and erythrocyte compartments suggests that the total glutathione (GSH+GSSG) deficit is due to heightened rate of GSH utilization rather than decreased synthesis capacity. While severity of erythrocyte GSH depletion was similar in SCD patients with and without PH, erythrocyte glutamine levels differed significantly (482±92μM, n=17 vs 934±134μM, n=23, p<0.02) and values inversely correlated to TRV (r = −0.51, p<0.0001). As glutamine is required for de novo synthesis of NAD(P)+ essential for GSH recycling, lower steady-state glutamine levels may reflect enhanced GSH utilization rates in the SCD erythrocytes. A significant reduction in the erythrocyte glutamine:glutamate ratio occurred in SCD patients compared with normal volunteers, with the lowest ratios observed in SCD patients with PH. The glutamine:glutamate ratio, potentially a gauge of NADPH biosynthesis and oxidative stress, was inversely correlated with TRV (r = −0.62, p<0.001), implicating glutamine bioavailability as a novel factor in the pathophysiology of PH. Changes in the glutamine:glutamate ratio were predominantly caused by decreased erythrocyte glutamine levels rather than increased glutamate levels, ruling out an effect on the ratio from increased cellular glutamine uptake. Erythrocyte glutamine:glutamate ratio correlated with age in patients with SCD (r = −0.33, p=0.04), and inversely correlated with plasma arginase concentratoin (r= −0.45, p=0.012), and plasma-Hb (r= −0.41, p=0.01), linking lower glutamine bioavailability to increased red cell derived plasma arginase, hemolysis and potentially with increased mortality in PH of SCD as previously reported (Morris et al, JAMA 2005). Decreased erythrocyte total glutathione and glutamine levels contribute to alterations in the erythrocyte redox environment, which compromise erythrocyte integrity and NO bioavailability and may play a role in hemolysis and the pathogenesis of PH of SCD.

Download Full-text

Nitric Oxide Effects in Sickle Cell Disease.

Blood ◽

10.1182/blood.v112.11.sci-48.sci-48 ◽

2008 ◽

Vol 112 (11) ◽

pp. sci-48-sci-48

Author(s):

Lori Styles

Keyword(s):

Nitric Oxide ◽

Pulmonary Hypertension ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Oxidant Stress ◽

Cell Disease ◽

No Donors ◽

Skin Ulcers ◽

No Bioavailability ◽

Inhaled No

Sickle cell disease (SCD) is a complex hemoglobinopathy characterized by microvascular occlusion and hemolytic anemia. Patients suffer from a myriad of both acute and chronic problems affecting virtually every organ system. Historically, microvascular occlusion has been the focus of scientific investigations into these manifestations and the chronic hemolysis of SCD was overlooked. More recently, however, the importance of the pathophysiology of hemolysis has been appreciated and related to a subset of the clinical manifestations of SCD, including pulmonary hypertension, priapism, skin ulcers, and possibly stroke. This subphenotype of SCD has been convincingly related to impaired nitric oxide (NO) homeostasis due to hemolysis. NO has pleiotropic effects including vaso-dilatory, antioxidative, anti-adhesion, and anti-thrombotic properties, which are all potentially important in the pathophysiology of SCD. Perturbation of NO homeostasis, therefore, could profoundly impact patients with SCD. Animal and human data support a state of “NO resistance” in SCD patients. Human studies have shown that SCD patients have a decreased response to exogenous NO donors and that is likely due to the scavenging of NO by free plasma hemoglobin that results from ongoing hemolysis. “NO resistance” is further augmented by the increased levels of reactive oxygen species (ROS) known to occur in SCD patients. High levels of ROS favor additional hemolysis through increased oxidant stress on the sickle red blood cell and reduce NO bioavailability by inactivation of circulating NO. With the substantial human and animal data to support a role for “NO resistance” in the pathophysiology of SCD, investigation with NO-based therapy have begun. Several approaches to overcoming “NO resistance” can be devised including increasing the precursors to NO, decreasing hemolysis, direct NO donors, and decreasing oxidant stress. To date, studies evaluating arginine (NO precursor), inhaled NO, and sildenafil (NO donor) have been reported. Oral arginine showed no benefit in a large clinical trial, and a preliminary trial of inhaled NO had only minimal benefit. Sildenafil may be more promising and is under further study. Lastly, although impaired NO bioavailability has been related to a subset of patients with pulmonary hypertension, skin ulcers and priapism, it will be important to determine what impact NO has on other manifestations, such as vaso-occlusive pain episodes and whether NO modulation can also be used therapeutically in this setting.

Download Full-text

Cardiopulmonary Complications of Sickle Cell Disease: Role of Nitric Oxide and Hemolytic Anemia

Hematology ◽

10.1182/asheducation-2005.1.51 ◽

2005 ◽

Vol 2005 (1) ◽

pp. 51-57 ◽

Cited By ~ 57

Author(s):

Mark T. Gladwin ◽

Gregory J. Kato

Keyword(s):

Nitric Oxide ◽

Pulmonary Hypertension ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Iron Chelation ◽

Cell Disease ◽

Intravascular Hemolysis ◽

Inflammatory Stress ◽

Risk Of Death ◽

Cardiopulmonary Complications

Abstract Medical advances in the management of patients with sickle cell disease, thalassemia, and other hemolytic anemias have led to significant increases in life expectancy. Improved public health, neonatal screening, parental and patient education, advances in red cell transfusion medicine, iron chelation therapy, penicillin prophylaxis for children, pneumococcal immunization, and hydroxyurea therapy have all likely contributed to this effect on longevity.1,2 Importantly, as a generation of patients with sickle cell disease and thalassemia ages, new chronic complications of these hemoglobinopathies develop. In this context, pulmonary hypertension is emerging as one of the leading causes of morbidity and mortality in adult sickle cell and thalassemia patients, and likely in patients with other hemolytic anemias. A common feature of both sickle cell disease and thalassemia is intravascular hemolysis and chronic anemia. Recent data suggest that chronic intravascular hemolysis is associated with a state of endothelial dysfunction characterized by reduced nitric oxide (NO) bioavailability, pro-oxidant and pro-inflammatory stress and coagulopathy, leading to vasomotor instability and ultimately producing a proliferative vasculopathy, a hallmark of which is the development of pulmonary hypertension in adulthood.3–5 In conclusion, pulmonary hypertension is common in patients with hereditary hemolytic anemias and is associated with a high risk of death in patients with sickle cell disease. New therapies targeting this vasculopathy and aimed at normalizing the vasodilator:vasoconstrictor balance are discussed.

Download Full-text

Lactate dehydrogenase as a biomarker of hemolysis-associated nitric oxide resistance, priapism, leg ulceration, pulmonary hypertension, and death in patients with sickle cell disease

Blood ◽

10.1182/blood-2005-06-2373 ◽

2006 ◽

Vol 107 (6) ◽

pp. 2279-2285 ◽

Cited By ~ 375

Author(s):

Gregory J. Kato ◽

Vicki McGowan ◽

Roberto F. Machado ◽

Jane A. Little ◽

James Taylor ◽

...

Keyword(s):

Nitric Oxide ◽

Pulmonary Hypertension ◽

Lactate Dehydrogenase ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Serum Lactate Dehydrogenase ◽

Cell Disease ◽

Intravascular Hemolysis ◽

Soluble Adhesion Molecules ◽

Risk Of Death

AbstractPulmonary hypertension is prevalent in adult patients with sickle cell disease and is strongly associated with early mortality and markers of hemolysis, in particular, serum lactate dehydrogenase (LDH). Intravascular hemolysis leads to impaired bioavailability of nitric oxide (NO), mediated by NO scavenging by plasma oxyhemoglobin and by arginine degradation by plasma arginase. We hypothesized that serum LDH may represent a convenient biomarker of intravascular hemolysis and NO bioavailability, characterizing a clinical subphenotype of hemolysis-associated vasculopathy. In a cohort of 213 patients with sickle cell disease, we found statistically significant associations of steady-state LDH with low levels of hemoglobin and haptoglobin and high levels of reticulocytes, bilirubin, plasma hemoglobin, aspartate aminotransferase, arginase, and soluble adhesion molecules. LDH isoenzyme fractionation confirmed predominance of LD1 and LD2, the principal isoforms within erythrocytes. In a subgroup, LDH levels closely correlated with plasma cell-free hemoglobin, accelerated NO consumption by plasma, and impaired vasodilatory responses to an NO donor. Remarkably, this simple biomarker was associated with a clinical subphenotype of pulmonary hypertension, leg ulceration, priapism, and risk of death in patients with sickle cell disease. We propose that LDH elevation identifies patients with a syndrome of hemolysis-associated NO resistance, endothelial dysfunction, and end-organ vasculopathy.

Download Full-text

Lactate Dehydrogenase as a Biomarker of Hemolysis-Associated Nitric Oxide Resistance, Priapism, Leg Ulceration, Pulmonary Hypertension and Death in Patients with Sickle Cell Disease.

Blood ◽

10.1182/blood.v106.11.3188.3188 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3188-3188 ◽

Cited By ~ 1

Author(s):

Gregory J. Kato ◽

Vicki McGowan ◽

Roberto F. Machado ◽

Jane A. Little ◽

James Taylor ◽

...

Keyword(s):

Nitric Oxide ◽

Pulmonary Hypertension ◽

Lactate Dehydrogenase ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Serum Lactate Dehydrogenase ◽

Cell Disease ◽

Intravascular Hemolysis ◽

Soluble Adhesion Molecules ◽

Risk Of Death

Abstract Pulmonary hypertension is prevalent in adult patients with sickle cell disease, and is strongly associated with markers of hemolysis, in particular serum lactate dehydrogenase (LDH), and early mortality. Intravascular hemolysis leads to a state of resistance to nitric oxide (NO), mediated by NO scavenging by plasma oxyhemoglobin and by arginine degradation by plasma arginase. We hypothesized that serum LDH may represent a convenient biomarker of intravascular hemolysis and NO bioavailability, characterizing a clinical subphenotype of hemolysis-associated vasculopathy. In a cohort of 213 patients with sickle cell disease, we found statistically significant associations of LDH with low levels of hemoglobin and haptoglobin and high levels of reticulocytes, bilirubin, plasma hemoglobin, aspartate aminotransferase, arginase and soluble adhesion molecules. LDH isoenzyme fractionation confirmed predominance of LD1 and LD2, the principal isoforms within erythrocytes. In a subgroup, LDH levels closely correlated with plasma cell free hemoglobin, accelerated nitric oxide consumption by plasma and impaired vasodilatory responses to an NO donor. Remarkably, this simple biomarker was statistically associated with a clinical subphenotype of pulmonary hypertension, leg ulceration, priapism, and risk of death in patients with sickle cell disease. We propose that LDH elevation identifies patients with a syndrome of hemolysis-associated NO-resistance, endothelial dysfunction and end-organ vasculopathy. Figure Figure Figure Figure

Download Full-text

Mutations and Polymorphisms Influencing Hemolysis in Hemoglobin Genes and Risk of Pulmonary Hypertension in Sickle Cell Disease: Effect of Hemoglobin SC.

Blood ◽

10.1182/blood.v108.11.1206.1206 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1206-1206

Author(s):

James G. Taylor ◽

Diana Ackah ◽

Crystal Cobb ◽

Oswaldo Castro ◽

Gregory J. Kato ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Survival Data ◽

Strong Predictor ◽

Pulmonary Vasculature ◽

Cell Disease ◽

Intravascular Hemolysis ◽

Hepatic Congestion ◽

Significant Difference

Abstract Pulmonary hypertension (PH) is an increasingly recognized phenotypic manifestation of sickle cell disease (SCD) and a leading cause of death among adults with SCD. Chronic intravascular hemolysis is a central pathologic event with release of hemoglobin into plasma and leads to nitric oxide consumption. A resulting NO deficiency state is hypothesized to produce vasoconstriction within the pulmonary vasculature, ultimately becoming manifest as symptomatic PH. We have hypothesized that genetic factors may influence the risk for developing PH in SCD. As an initial step in identifying PH susceptibility loci, the allelic spectrum of mutations and polymorphisms in the hemoglobin genes have been examined with respect to PH in adults with SCD. Echocardiography was used to phenotype 261 unrelated subjects for PH, where cases were prospectively defined by a triscuspid regurgitant jet velocity > 2.5 m/s. The entire HBB gene was sequenced, and genotypes for SNPs flanking the β and α-globin loci were determined. HBB sequence analysis identified 66 (25.3%) compound heterozygotes for 1 of 13 different mutations (16.1% Hb SC or 9.2% β+ thalassemia, β0 thalassemia, or another hemoglobinopathy). A significant difference in genotype distributions between PH cases and cohort controls (3 x 2 table, P=0.026) suggested that individual mutations within the locus may be associated with PH. When analyzed individually, the prevalence of HbSC between cases (7.4%) and controls (21.1%) suggested that the HbC allele is associated with protection from PH (Odds Ratio=0.35, 95% CI 0.15–0.78, P=0.009). HbSS is only a mild risk factor (OR=2.01, 95% CI 1.07–3.78, P=0.026). In contrast, there was no association between 5′ or 3′ β globin locus haplotypes defined by combinations of 16 common SNPs and PH in patients with either SCD or HbSS. When the α-globin locus was examined using 3 haplotype tagged SNPs flanking the HBA2 and HBA1 genes, no PH associations were observed. An analysis of α-thalassemia mutations has not yet been completed, thus no definitive conclusions can be made for the influence of the α-globin locus on PH. Finally, the HbSC association was explored further by comparing laboratory markers of hemolysis and survival data within the HbSC group. Between SC PH cases and controls, anemia was the only significant characteristic of PH (P=0.0027) without other labs suggestive of excessive hemolysis. In contrast, other clinical findings observed in association with SCD PH, including hepatic congestion, iron overload and renal insufficiency, were present in HbSC PH group. While an overall association with protection from PH was observed for HbSC, a preliminary 40 month survival analysis suggests that PH in SC patients remains a significant marker associated with a risk for death (Hazard Ratio=7.89, P=0.007) which is comparable to overall SCD survival with PH. We conclude that the HbSC genotype is associated with protection from the development of PH in SCD, possibly due a smaller relative contribution of intravascular hemolysis. However, this diagnosis remains a strong predictor for death in SCD independent of HBB genotype. Further study of large populations of HbSC patients are warranted for defining optimal PH therapies and may be useful for elucidating the genetic basis for the non-hemolytic mechanisms contributing to secondary PH in SCD.

Download Full-text