Role of the human erythrocyte in generation and storage of asymmetric dimethylarginine

Davids M, van Hell AJ, Visser M, Nijveldt RJ, van Leeuwen PAM, Teerlink T. Role of the human erythrocyte in generation and storage of asymmetric dimethylarginine. Am J Physiol Heart Circ Physiol 302: H1762–H1770, 2012. First published February 24, 2012; doi:10.1152/ajpheart.01205.2011.—Proteolytic activity in whole blood may lead to release of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA). We investigated the role of the human erythrocyte in storage and generation of ADMA in healthy controls (n 36) and critically ill patients (n 38). Both free and total (sum of free and protein-incorporated) ADMA were measured. Upon incubation of intact erythrocytes with extracellular ADMA (0 to 40 mol/l), equilibrium between intraand extracellular ADMA was reached within 3 h. Compared with controls, patients had significantly higher basal concentrations of ADMA in plasma (0.88 0.75 vs. 0.41 0.07 mol/l) and erythrocytes (1.28 0.55 vs. 0.57 0.14 mol/l). Intracellular and plasma ADMA were significantly correlated in the patient group only (r 0.834). Upon lysis, followed by incubation at 37°C for 2 h, free ADMA increased sevenfold (to 8.60 3.61 mol/l in patients and 3.90 0.78 mol/l in controls). In lysates of controls, free ADMA increased further to 9.85 1.35 mol/l after 18 h. Total ADMA was 15.43 2.44 mol/l and did not change during incubation. The increase of free ADMA during incubation corresponded to substantial release of ADMA from the erythrocytic protein-incorporated pool (21.9 4.6% at 2 h and 60.8 7.6% at 18 h). ADMA was released from proteins other than hemoglobin, which only occurred after complete lysis and was blocked by combined inhibition of proteasomal and protease activity. Neither intact nor lysed erythrocytes mediated degradation of free ADMA. We conclude that intact erythrocytes play an important role in storage of ADMA, whereas upon erythrocyte lysis large amounts of free ADMA are generated by proteolysis of methylated proteins, which may affect plasma levels in hemolysis-associated diseases.