Carbamylation of erythrocyte membrane aminophospholipids: an in vitro and in vivo study

Objectives: To study the binding of cyanate to erythrocyte membrane aminophospholipids in vitro, and to investigate whether carbamylated aminophospholipids can be detected in the plasma membrane of native erythrocytes. Design and Methods: For in vitro studies, the lipid components of 14C-carbamylated erythrocyte membranes were resolved by thin-layer chromatography (TLC). The covalent incorporation of cyanate was visualized by autoradiography and quantitated by phosphorus analysis. For the in vivo studies, phospholipid headgroups were enzymatically hydrolyzed by phospholipase D and subsequently reacted with diacetyl monoxime. Results: Both phosphatidylethanolamine (PE) and phosphatidylserine (PS) were covalently modified by [14C] cyanate; incorporating 15.76 ± 0.09 and 13.34 ± 0.81 mol%, respectively, following a 15-h incubation. Carbamylated PE (carb-PE) was resolved with PE by TLC in a solvent system consisting of chloroform/methanol/ammonia (65/35/5, v/v/v). Treatment of native erythrocyte membrane lipid micelles with phospholipase D, followed by reaction with diacetyl monoxime, suggests the presence of intrinsic carb-PE (2.85 ± 0.65 percent of the total PE). Conclusions: Carbamylation of erythrocyte aminophospholipid may be involved in some of the hematological consequences of uremia on the erythrocyte