D 2500

The HPLC apparatus was comprised of a HITACHI ELITE LaChrom system (L2130) equipped with a Hitachi L-3000 detector and a D-2500 chromato-integrator. Peptides were purified by RP-HPLC using a Cosmosil 5C₁₈-AR-II column (4.6 × 150 mm, Nacalai tesque Inc., Kyoto, Japan). The peptides were separated by a linear gradient of CH₃CN in 0.05% TFA increasing at a rate of 1%/min from solvent A (0.05% TFA/H₂O) to solvent B (0.05% TFA/CH₃CN) at a flow rate of 1 mL/min [8 (link),21 (link)].

The employed HPLC units were:−

Chiral HPLC analysis: a Merck-Hitachi (Hitachi Ltd., Tokyo, Japan), equipped with a UV detector model L-4250, pump system model L-6200 and a chromato-integrator model D-2500. The column employed in the analyses was a Phenomenex Lux-Cellulose 1 (Phenomenex, Torrance, CA, USA). The dimension of the column is 250 mm × 4.6 mm, 3 µm. The elution was in isocratic mode with the indicated eluant and flow. All the samples were measured at λ = 254 nm and 25 °C.

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RP-HPLC analysis: Agilent 1100 system (Agilent Technologies, Waldbronn, Germany) equipped with a Zorbax SB-C18 column (150 mm × 3.0 mm, 3.5 µm) for 1 and with a Supelco Discovery C18 (250 mm × 4.6 mm, 5.0 µm) for 7.

All the samples were measured at λ = 254 nm and 25 °C.

To check absorption of fatty acids by mice, the fatty acid composition of plasma lipids was assessed by gas chromatography. Plasma total lipids were extracted by the method of Bligh and Dyer with chloroform: methanol (2:1, v:v). Methyl esters of fatty acids were obtained by transesterification with boron trifluoride in methanol at 80 °C for 60 min. An aliquot containing the methyl esters of fatty acids was analyzed by gas chromatography (Hewlett-Packard model 5890) on a 30 m RTX-2330 column (Restek, Bellefonte, PA, USA) with an internal diameter of 0.25 mm equipped with a flame ionization detector. The carrier gas was helium at a pressure of 105 kPa. For the total separation of the different compounds, two temperature settings were established: 140 °C–200 °C at 3 °C/min or in two stages of 140 °C–180 °C at 4 °C/min and 180 °C–210 °C at 2 °C/min. The temperature of the injector and detector was 260 °C. The linear response of the detector was tested periodically with standard mixtures. Typically, two internal standards with different molecular weight (13:0 and 23:0 or 27:0) were used. Peaks were integrated with an integrator D-2500 (Hitachi Ltd., Tokyo, Japan) and identified by comparison of retention times with standards. When necessary, the identification may be confirmed by mass spectrometry (Hewlett-Packard detector model 5970B) at an ionization potential of 70 eV.