Elevated Plasma Arginase Levels in Hemoglobinopathies.

Abstract

The decreased bioavailability of arginine (Arg) and the resulting lower nitric oxide (NO) production has been shown to be an important factor in the pathology of sickle cell disease (SCD) and thalassemia. Vascular alterations leading to pulmonary hypertension are important factors of heart failure and death in these hemoglobinopathies. Red blood cell (RBC) hemolysis in these patients will release arginase into the circulation and contribute to the reduction of plasma Arg levels, change the Arg-to-ornithine ratio, and increase other downstream amino acid metabolites. Such compounds, including proline and polyamines, may contribute to vascular and airway remodeling. To study the contribution of arginase released from RBC, we measured the arginase activity and arginase protein concentration in the plasma and RBC lysates of normal controls, SCD patients, and thalassemia patients.Arginase activity was determined by the conversion of 14C-labeled guanidine-L-arginine to 14C-labeled urea, then to 14CO2 by urease and trapped as Na214CO3 for scintillation counting. Arginase and hemoglobin concentration were measured by ELISA (BioVendor, Candler, NC and Bethyl Labs Inc., Montgomery, TX respectively). Mean cellular hemoglobin concentration was measured with a Coulter Counter (Beckman Coulter, Fullerton, CA) or Technicon H3 analyzer (Tarrytown, NY). RBC were separated by density gradient centrifugation with OptiPrep (Axis Shield, Dundee, UK). Arginase specific activity (SA) was calculated by dividing the activity by the concentration to yield mole of Arg metabolized per g of arginase per hour. We found a higher level of arginase in the plasma and RBC of SCD and thalassemia patients as compared to normal controls. These levels correlated with cell-free hemoglobin content (plasma) or reticulocyte count (RBC). The light density fraction (<1.077 g/ml) of RBC, enriched in reticulocytes, showed higher arginase levels confirming increased arginase levels in young RBC. Interestingly, as the plasma arginase protein and activity increased with cell-free hemoglobin, the SA decreased. Similarly in RBC lysates, the SA decreases with hemoglobin concentration. This suggests that a factor exists within the RBC that negatively modulates arginase activity. Due to the high SA of arginase at low hemoglobin concentrations, low levels of intravascular hemolysis can generate relatively high levels of Arg breakdown.We conclude that low levels of intravascular hemolysis of RBC with increased arginase content in SCD and thalassemia, play a role in the altered Arg metabolism and decreased NO production. While the reason for high levels of arginase in the RBC as well as complete clinical impact of elevated arginase activity in plasma remain to be determined, dysregulated Arg metabolism and a shift toward Arg catabolic products, may be associated with the development of pulmonary hypertension and mortality in SCD and thalassemia.