Dysregulated arginine metabolism, hemolysis-associated pulmonary hypertension, and mortality in sickle cell disease

Abstract

Context: Sickle cell disease is characterized by a state of nitric oxide resistance and limited bioavailability of l-arginine, the substrate for nitric oxide synthesis. We hypothesized that increased arginase activity and dysregulated arginine metabolism contribute to endothelial dysfunction, pulmonary hypertension, and patient outcomes.

Objective: To explore the role of arginase in sickle cell disease pathogenesis, pulmonary hypertension, and mortality.

Design: Plasma amino acid levels, plasma and erythrocyte arginase activities, and pulmonary hypertension status as measured by Doppler echocardiogram were prospectively obtained in outpatients with sickle cell disease. Patients were followed up for survival up to 49 months.

Setting: Urban tertiary care center and community clinics in the United States between February 2001 and March 2005.

Participants: Two hundred twenty-eight patients with sickle cell disease, aged 18 to 74 years, and 36 control participants.

Main outcome measures: Plasma amino acid levels, plasma and erythrocyte arginase activities, diagnosis of pulmonary hypertension, and mortality.

Results: Plasma arginase activity was significantly elevated in patients with sickle cell disease, with highest activity found in patients with secondary pulmonary hypertension. Arginase activity correlated with the arginine-ornithine ratio, and lower ratios were associated with greater severity of pulmonary hypertension and with mortality in this population (risk ratio, 2.5; 95% confidence interval [CI], 1.2-5.2; P = .006). Global arginine bioavailability, characterized by the ratio of arginine to ornithine plus citrulline, was also strongly associated with mortality (risk ratio, 3.6; 95% CI, 1.5-8.3; P<.001). Increased plasma arginase activity was correlated with increased intravascular hemolytic rate and, to a lesser extent, with markers of inflammation and soluble adhesion molecule levels.

Conclusions: These data support a novel mechanism of disease in which hemolysis contributes to reduced nitric oxide bioavailability and endothelial dysfunction via release of erythrocyte arginase, which limits arginine bioavailability, and release of erythrocyte hemoglobin, which scavenges nitric oxide. The ratios of arginine to ornithine and arginine to ornithine plus citrulline are independently associated with pulmonary hypertension and increased mortality in patients with sickle cell disease.

Figures

Figure 1. The arginine-to-ornithine ratio as a surrogate marker for arginase activity, its association with pulmonary hypertension (PHT) and correlation with arginase activity

A. The arginine-to-ornithine ratio (Arg/Orn) in normal controls (Control, n=36) vs. patients with SCD (n=209). B. Arginine-to-ornithine ratio in sickle cell patients with a tricuspid regurgitant jet velocity (TRV) by echocardiography 2.5-2.9 m/s (mild PHT) vs. patients with a TRV ≥ 3.0 m/s (severe PHT). C. Plasma arginase activity (μmol/ml/hr) in normal controls (Control, n=36) vs. patients with sickle cell disease (SCD, n=140). D. Correlation of plasma arginase activity to the arginine-to-ornithine ratio.

Figure 2. Association of arginase activity with hemolytic rate

Correlation of plasma arginase activity (μmol/ml/hr) to cell-free hemoglobin (Cell-Free Hb, n=138, p< 0 .001) in patients with sickle cell disease.

Figure 3. Arginase activity in red blood cell lysate vs. plasma

A. Red blood cell (RBC)–lysate arginase activity (nmol/mg/min) in normal controls (Control, n=45) compared to patients with sickle cell disease (SCD, n=16). B. Correlation of plasma arginase to red blood cell-lysate arginase activity in control patients (black circles) and patients with sickle cell disease (red circles; r=0.43, p< 0.001). For purposes of comparison, horizontal and vertical dotted lines are set at approximately the 80th percentile for arginase activities of RBC-lysates and plasma, respectively, for control patients.

Figure 4. Association of arginine bioavailability ratios with mortality in sickle cell disease: Kaplan-Meier survival plots

A. Survival for three categories of Arginine-to-Ornithine ratio: “High” = upper quartile, > 0.8690; “Medium” = 25th to 75th percentiles, > 0.4385 and ≤ 0.8690; “Low” = lower quartile, ≤ 0.4385. B. Survival for three categories of Arginine-to-(Ornithine + Citrulline) ratio: “High” = upper quartile, > 0.6254, “Medium” = 25th to 75th percentiles, > 0.3245 and ≤ 0.6254, “Low” = lower quartile, ≤ 0.3245.

Figure 5. Altered arginine metabolism in sickle cell disease

Arginine is synthesized endogenously primarily via the intestinal-renal axis . Stimuli that increase activities of nitric oxide synthase (NOS) and arginase are indicated, as are conditions that result in increased NO consumption. Potential consequences of elevated ornithine production are also indicated.