الفهرس 
AcknowledgmentOnly 14 pages are availabe for public view
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Abstract
This study was conducted on 35 normal healthy volunteer donors (15 normal healthy donors for plasma collection and another 20 healthy donors for RBCs collection) coming to the transfusion unit. All donors gave informed consent before being included in the study. The following was done: Lipoprotein preparation, isolation and oxidation: 🞭 Plasma collection and preparation: Plasma collection was done on 15 healthy normolipidemic donors after overnight fast through apharesis using Spectra Optia Apharesis System. 🞭 Density gradient ultracentrifugation for LDL isolation : LDL isolation from re-calcified plasma (by adding 3.675 g/L calcium chloride) followed by density gradient ultracentrifugation (using solid potassium bromide 0.019 g/mL and Thromborel-S Siemens 0.1 m/L concentration) for separation and removal of VLDL and IDL. 🞭 Dialysis and ultrafiltraion of LDL: The LDL fraction was collected and simultaneously dialyzed several times (in PBS solution Gibco) through a collodium bag (Servapor dialysis tubing, 29 mm diameter). Thereafter, the LDL solution was filtered through sterile vacuum filtration 50 mL Falcon tube (Millipore Steriflip Vacuum-drive Filtration System). 🞭 Oxidation of LDL: The in vitro oxidation of LDL was performed by adding freshly prepared 93.6 uL of 4% CuSO4 during dialysis for 40-60 hour in dark at 4°C till complete decolourization occurs. This LDL and/or oxLDL were valid for only two weeks. RBCs Preparation: 50 mL of peripheral blood, from 20 healthy donors, obtained by standard cubital venipuncture (without mechanical hemolysis of blood cells), then washed, pelleted and be ready for cultivation with different concentration of nLDL and oxLDL and at different time points. - Erythrocytic pellets: Erythrocytic pellets were analysed by mass spectrometery GCMS to evaluate the oxysterol content of RBCs (7-hydroxycholesterol, 7- ketocholesterol and 25-hydroxycholesterol) and by flow cytometery to evaluate the release of RBC-EVs (Annexin V- 7AAD Panel) and for detection of lipid associated-raft and erythrocytic plasma membrane microdomains (CD55, CD47, CD147 and CD59).– Supernatant collected: Supernatants were collected in Eppendorf cups for (lactate concentration and lactate dehydrogenase activity) to asses the storage lesion and start of apoptosis and flow cytometeric measurements to asses release of RBC-EVs in supernatant (CD235a). Finding of this study: RBCs intercalate sterols from lipoproteins There were significantly higher levels of 7-hydroxycholesterol, 7- ketocholesterol and 25-hydroxycholesterol in oxLDL-treated RBCs at all concentrations compared to nLDL treated RBCs. Quantification of the oxysterol content of RBCs after incubation with 200 ug/mL concentration of nLDL and oxLDL at different time points RBCs treated oxLDL at different time points (from 20 mintues to 24 hours) showed statistically significant increases in levels of oxysterols in comparison to RBCs loaded with nLDL. Longer incubation of RBCs with oxLDL did not result in more folds increase in cellular oxysterol contents in comparison to nLDL-treated RBCs. RBCs respond to nLDL and oxLDL by differential release of lactate and LDH Lactate concentrations in collected supernatants were significantly raised after 6 hours of incubation with both nLDL and oxLDL in comparison with earlier hours of incubation (at 40 minutes of incubation). Lactate Dehydrogenase (LDH) activities were relatively constant through the first few hours of incubation (estimated at 40 minutes, 80 minutes, 2 hours and 6 hours). At the 16 hour, 1.6- and 1.4-folds increased were observed for oxLDL and nLDL respectively, while 2.1- and 2.2-folds were shown at 24 hour for both oxLDL and nLDL respectively. Lipoproteins induce differential extent of eryptosis and release of RBC-EVs Cultured RBCs incubated with oxLDL at a concentration of 200 ug/mL showed a higher expression of Annexin-V positive/CD235a-positive RBCs, with a prominant impact at 2 hours, 6 hours and 16 hours of incubation compared to those incubated with the same concentration of nLDL at the same time periods. Cytoflourimeteric measurement of extracellular vesicles of cultured RBCs secreted in supernatant Our current data shows no significant difference in the amount of EV’s release from erythrocytes incubated with both nLDL and oxLDL at concentration of 200 ug/mL. Detergent membrane solubility of erythrocytic surface antigens CD55, CD59, CD47 and CD147 displayed no significant difference in the TX100-solubility; hence showed the FCDR indexes no difference between nLDL and oxLDL in comparison to 1% BSA (negative control) at different time points and concentration and no significant difference between nLDL and oxLDL.