(11c) Quantitative Analysis of the Phospholipid Composition of the Red Blood Cell Membrane Using Cyclodextrins

The phospholipid bilayer membrane around mammalian cells is known to be asymmetric in type and composition of phospholipids. However, due to the characteristics of a lipid bilayer, efforts to measure the phospholipid composition of each leaflet, without disturbing the other have fallen short. Our work implements phospholipid exchange using cyclodextrin to report the type and species of phospholipids in the outer leaflet of red blood cells (RBCs).

The outer leaflet of RBCs was extracted through an exchange process with exogenous phospholipids. Methyl-α-cyclodextrin was used to mediate the exchange, as its hydrophobic cavity is large enough to form inclusion complexes with acyl chains of phospholipids, but not with cholesterol. Different parameters of the exchange process were optimized to obtain the lowest hemolysis, as it would indicate lysis of RBCs and inner leaflet contamination in the extracted lipids. Fluorescence microscopy was used to confirm the delivery of a fluorescent lipid to the membrane and a quenching agent was used to examine the presence of fluorescent phospholipids in the inner leaflet. The extracted lipids were then analyzed using thin layer chromatography to show the most abundant phospholipids in the outer leaflet of the membrane. Lipidomic analysis was performed on total lipid extractions of RBCs after exchange with a phospholipid that is not naturally available in RBCs. Analysis revealed phosphatidylcholines and sphingomyelins are the most abundant phospholipids in the RBC outer leaflet with 34:1 PC and 16:0 SM as the most abundant acyl chain saturations.

Senescent RBCs are known to have phosphatidylserine in their outer leaflet, and this is linked with their clearance through macrophages. We examined whether lipid exchange can be used to reconstitute the outer leaflet of senescent RBCs, and remove the phosphatidylserine. Senescent RBCs showed no phosphatidylserine on their outer leaflet after exchange and in vitro macrophage clearance was extremely decreased.