(176y) Quantitative Analysis of the Plasma Membrane Outer Leaflet in Red Blood Cells

The plasma membrane of mammalian cells is a lipid bilayer that is asymmetric with respect to its phospholipid composition. The phospholipid composition of lipids in the inner leaflet (facing the cytoplasm) is different from the outer leaflet (facing the outside environment). Membrane lipid asymmetry plays an important role in various cellular functions such as apoptosis and activation of phagocytes and platelets. While it is well known that cells actively maintain phospholipid asymmetry, the exact lipid composition of each leaflet of the bilayer is still not known for many cells. This is due to the nature of a lipid bilayer, making it difficult to characterize one leaflet without disturbing the other. The current work aims to develop a simple method for quantitative analysis of the outer leaflet of the plasma membrane in mammalian cells with a focus on human red blood cells for which the outer leaflet is characterized, allowing for the validation of the method.

The lipids in the outer leaflet of red blood cells were extracted using methyl-α-cyclodextrin (MαCD), a cyclic molecule with a hydrophobic cavity. To prevent red blood cell disruption and hemolysis during the extraction of the outer leaflet, MαCD was pre-loaded with exogenous sphingomyelin (SM), phosphatidylcholine (POPC), phosphoethanolamine (POPE), and phosphatidylserine (POPS) at four different concentrations between 0.375 to 3 mM. Lipid-loaded MαCD was incubated with the cells for three time steps to allow the lipid exchange to proceed. The exchange process facilitates replacing the lipids in the outer leaflet of the red blood cells with the exogenous lipids loaded in MαCD. Delivery of the lipids to the membrane was examined using a fluorescent lipid (NBD-PE) and confocal microscopy. Red blood cells’ hemolysis was measured after the exchange using UV absorbance at 541 nm. Thin Layer Chromatography (TLC) was used to identify the extracted lipids. Lipidomics analysis using mass spectroscopy (MS) was performed to quantify the level of each extracted lipid.

Addition of MαCD led to a significant amount of hemolysis; however, preloading MαCD prevented this hemolysis depending on the composition and concentration of the loaded lipids. The results indicated that POPE at all concentrations, SM at high concentrations, and POPS at low concentrations caused hemolysis, but POPC did not cause cell disruption. Lipids extracted by MaCD were characterized using TLC in treatments for which no hemolysis was observed. The TLC plate showed bands for SM and POPC only, which is in complete agreement to the known composition of outer leaflet of red blood cells. MS confirms the lipid compositions of SM and POPC and trace amounts of other lipids. This simple and novel method can be used to analyze the outer leaflet membrane composition in different cells or in diseased vs. healthy cells.