Palmitic acid

Cyclohexanol (>98.0%) and cis-3-hexen-1-ol (97.0 + %) were obtained from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). Palmitic acid was acquired from Nacalai Tesque, Inc. (Kyoto, Japan). trans-2-Decenal (>93.0%), trans-2-heptenal (>95.0%), trans-2-hexen-1-ol (>95.0%), trans-2-hexenal (>97.0%), cis-3-hexenyl acetate (>97.0%), and hexyl acetate (>99.0%) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The other reagents were described in our previous paper.^{11 (link)} The impurities derived from extraction solvents (hexane, methanol, and dichloromethane) for OA-LLE, SAFE, and HS-SPME were not observed. The monovarietal extra virgin olive oils (EVOO), Hojiblanca produced in Andalucía (Spain) and Mission and Lucca produced in Kagawa (Japan), were used in this study. These EVOOs were purchased from a market in Japan and stored at −20 °C until used.

Human dermal microvascular endothelial cells (Promo Cell, Heidelberg, Germany) were incubated in endothelial cell growth medium (Promo Cell). N/TERT-1 keratinocytes derived from normal human epidermal keratinocytes and immortalized with the telomerase catalytic subunit were used for experiments. Cells were cultured in keratinocyte serum-free medium (Life Technologies) supplemented with 0.2 ng/ml of epidermal growth factor (EGF), 25 mg/ml of bovine pituitary extract, 0.4 mM CaCl2 and penicillin/streptomycin. Both cells then incubated for 24 h in fresh growth medium containing 2% BSA in the absence or presence of 500 μmol/l palmitic acid, stearic acid, oleic acid and linoleic acid (Nacalai Tesque). These doses are well within the physiological range for free fatty acid concentrations reported for rodents and humans^{35 (link)}. Each fatty acid was dissolved in ethanol and diluted 1:100 in endothelial cell growth medium containing 2% (wt/vol) fatty acid–free BSA (Calbiochem, San Diego, Calif) before being added to the cells. Control cells received vehicle.