The erythrocyte fatty acid membrane profile analysis was carried out as previously described, using the erythrocyte membrane pellet obtained by standard methods [39] (link). For this study, selection of the erythrocyte fraction was made by modification of a literature procedure for the selection of aged erythrocytes (red blood cell age >3 months), with cells selected for high density and small diameter compared to the average erythrocyte population [40] .
One mL of whole blood was first centrifuged at 2000 g for 5 min to eliminate the plasma, and a second round of centrifugation was then carried out at 4000 g at 4°C for 5 min in order to yield a stratification by cell density. The bottom layer (2.5 mm from the bottom of tube) consisted of erythrocyte cells, which were evaluated for their diameter using a Scepter™ 2.0 Cell Counter (Merck Millipore, Milan, Italy) to characterize the cell selection from each blood sample. The results were also compared with the cell population obtained from standard density gradient separation [41] (link), [42] .
Briefly, lipids were extracted from erythrocyte membranes according to the method of Bligh and Dyer [43] . The phospholipid fraction was controlled by TLC as previously described [39] (link), then treated with KOH/MeOH solution (0.5 M) for 10 min at room temperature and under stirring [44] (link).
Fatty acid methyl esters (FAME) were extracted using n-hexane; the hexane phase was collected and dried with anhydrous Na2SO4. After filtration, the solvent was eliminated by evaporation using a rotating evaporator, and the thin white film of the FAME was subsequently dissolved in a small volume of n-hexane. Approximately 1 µL of this solution was injected into the GC. A Varian CP-3800 gas chromatograph, with a flame ionization detector and an Rtx-2330 column (90% biscyanopropyl-10% phenylcyanopropyl polysiloxane capillary column; 60 m, 0.25 mm i.d., 0.20 µm film thickness) was used for the analysis. Temperature was held at 165°C held for the initial 3 min, followed by an increase of 1°C/min up to 195°C, held for 40 min, followed by a second increase of 10°C/min up to 250°C, held for 5 min. The carrier gas was helium, held at a constant pressure of 29 psi. Methyl esters were identified by comparison with the retention times of commercially available standards or trans fatty acid references, obtained as described elsewhere [45] (link).
One mL of whole blood was first centrifuged at 2000 g for 5 min to eliminate the plasma, and a second round of centrifugation was then carried out at 4000 g at 4°C for 5 min in order to yield a stratification by cell density. The bottom layer (2.5 mm from the bottom of tube) consisted of erythrocyte cells, which were evaluated for their diameter using a Scepter™ 2.0 Cell Counter (Merck Millipore, Milan, Italy) to characterize the cell selection from each blood sample. The results were also compared with the cell population obtained from standard density gradient separation [41] (link), [42] .
Briefly, lipids were extracted from erythrocyte membranes according to the method of Bligh and Dyer [43] . The phospholipid fraction was controlled by TLC as previously described [39] (link), then treated with KOH/MeOH solution (0.5 M) for 10 min at room temperature and under stirring [44] (link).
Fatty acid methyl esters (FAME) were extracted using n-hexane; the hexane phase was collected and dried with anhydrous Na2SO4. After filtration, the solvent was eliminated by evaporation using a rotating evaporator, and the thin white film of the FAME was subsequently dissolved in a small volume of n-hexane. Approximately 1 µL of this solution was injected into the GC. A Varian CP-3800 gas chromatograph, with a flame ionization detector and an Rtx-2330 column (90% biscyanopropyl-10% phenylcyanopropyl polysiloxane capillary column; 60 m, 0.25 mm i.d., 0.20 µm film thickness) was used for the analysis. Temperature was held at 165°C held for the initial 3 min, followed by an increase of 1°C/min up to 195°C, held for 40 min, followed by a second increase of 10°C/min up to 250°C, held for 5 min. The carrier gas was helium, held at a constant pressure of 29 psi. Methyl esters were identified by comparison with the retention times of commercially available standards or trans fatty acid references, obtained as described elsewhere [45] (link).
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