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Plasma lipids were extracted by using the Matyash method with some modifications. Briefly, plasma (10 μL) was aliquoted in an Eppendorf tube containing the internal lipid standard mixture (40–400 ng/mL). Four hundred microliters of ice-cold 75% methanol with 0.1% butylated hydroxytoluene (BHT) was added to the plasma sample. Next, 1 mL MTBE was added, and the mixture was shaken for 1 h at room temperature. For phase separation, 250 μL of water was added, and centrifuged (14,000 x g, 4˚C, 15 min). The upper phase was transferred to a new tube, dried under vacuum and reconstituted in 100 μL chloroform/methanol (1:9, v/v).

Semi-quantitative lipid profiling was performed with reversed phase Kinetex C18 column (100 × 2.1 mm, 2.6 μm, Phenomenex, Torrance, CA, USA) for chromatographic separation of lipids. The mobile phase A consisted of water/methanol (1:9, v/v) containing 10 mM ammonium acetate, and the mobile phase B consisted of isopropanol/methanol (5:5, v/v) containing 10 mM ammonium acetate. The gradient elution program was as follows: 0 min (30% B), 0–15 min (95% B), 15–20 min (95% B), and 20–25 min (30% B). The flow rate was set a 200 μL/min. Five microliters of sample were injected for each run.

Semi-quantitative lipid profiling was performed by using a Nexera2 LC system (Shimadzu Corporation, Kyoto, Japan) connected to a triple quadrupole mass spectrometer (LC-MS 8040; Shimadzu, Kyoto, Japan) for chromatographic separation of lipids.