In situ assessment of erythrocyte membrane properties during cold storage

Membrane fluidity and overall protein secondary structure of human erythrocytes were studied in situ using Fourier transform infrared spectroscopy (FTIR). Erythrocyte membranes were found to have weakly cooperative phase transitions at 14degreesC and at 34degreesC, which were tentatively assigned to the melting of the inner membrane leaflet and the sphingolipid rich outer leaflet, respectively. Cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) resulted in a large increase in the cooperativity of these transitions, and led to the appearance of another phospholipid transition at 25degreesC. Multiple, sharp membrane phase transitions were observed after 5 days cold storage (4degreesC), which indicated phase separation of the membrane lipids. Using fluorescence microscopy, it was determined that the lipid probe 1, 1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (diI-C-18) remained homogeneously distributed in the erythrocyte membrane during cold storage, suggesting that lipid domains were below the resolution limit of the microscope. Using thin layer chromatography, changes in the membrane lipid composition were detected during cold storage. By contrast, assessment of the amide-II band with FTIR showed that the overall protein secondary structure of haemoglobin was stable during cold storage.