D J Trepanier1, R J Thibert. 1. Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Abstract
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.
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.
Authors: Tao Ji; Keisuke Ikehata; Yakov M Koen; Steven W Esch; Todd D Williams; Robert P Hanzlik Journal: Chem Res Toxicol Date: 2007-03-24 Impact factor: 3.739