Reagents and materials
RPMI-1640 medium without phenol red, cell culture freezing medium containing DMSO, fetal bovine serum (FBS), penicillin, streptomycin, 7.5% bovine serum albumin (BSA), phosphate buffer saline (PBS, pH = 7.4), and trypan blue were purchased from Life Technologies (Grand Island, NY). Rac-5,7-dimethyl tocol was purchased from Matreya (Pleasant Gap, PA). Alpha-tocopherol was obtained from Eastman (Kingsport, TN) and gamma-tocopherol was obtained Tama Biochemical Co. (Tokyo, Japan). Alpha-tocopheryl quinone and gamma-tocopheryl quinone were synthesized as described below.
RAW 264.7 murine macrophages (American Type Culture Collection, Rockville, MD) were cultured at 37°C in a humidified incubator (95 % air with 5% CO2) in RPMI-1640 medium with 10 % fetal bovine serum (FBS), 50 units/ml penicillin, and 50 mg/ml streptomycin (culture medium).
Enrichment of tocopherol (or tocopheryl quinone) in culture medium
A 50 μl aliquot of tocopherol (or tocopheryl quinone) solution in ethanol was placed in glass tube and dried under N2. After the addition of 1.0 ml of 7.5% BSA the mixture was vortexed for 2 min, 74.0 ml culture medium added, and the mixture vortexed again for 2 min. The final concentration of tocopherol (or tocopheryl quinone) in the culture medium was determined by HPLC (see below).
Uptake and depletion of tocopherols (or tocopheryl quinones) in cells
To define the time course of tocopherol (or tocopheryl quinone) accumulation, adherent cells (2 × 106/well) were subcultured in 12 well falcon tissue culture plates with medium enriched with about 5 μM tocopherol (or about 0.5 μM quinone) (3 ml/well). After 6 hr (the uptake phase), the medium was replaced with fresh medium without tocopherol (or quinone) and the depletion of tocopherols (or quinones) followed for an additional 4 hours (the depletion phase). During the course of the cell culture experiment, a 400 μl aliquot of medium was removed every 2 hr and placed in a glass vial for tocopherol (or quinone) measurement and the remaining medium removed. The adherent cells in the well were washed three times with 1.0 ml of PBS, and the cells overlaid with 400 μl of lysis buffer (1% Triton X-100 in pH 7.5, 20 mM HEPES buffer with 0.1 mM EDTA), scraped, and collected into a glass vial for tocopherol analysis by HPLC, and for protein determination by the BCA protein assay (Pierce, Rockford, IL). For each experiment, three wells were used per time point and each experiment was performed in duplicate. Statistical comparisons were made by use of the Student's t-test.
Tocopherol (or tocopheryl quinone) measurement by HPLC with electrochemical detection
The medium sample (or cell sample) was mixed with 1.0 ml ethanol and 1.0 ml hexane containing 10 μg/ml 2,6-di-tert-butyl-4-methylphenol (BHT), and rac-5,7-dimethyl tocol as an internal standard. The rac-5,7-dimethyl tocol internal standard was found to be very useful since it did not elute in the same position as gamma-tocopheryl quinone as does tocol (2-methyl-2-(4,8,12-trimethyltridecyl)chroman-6-ol) which is typically used as an internal standard. After vortexing, the mixture was centrifuged at 1020 × g for 5 min, and a 400 μl aliquot of the upper layer removed, dried under N2, and taken up in 100 μl of mobile phase. The mobile phase was methanol: water (90:10) containing 3 FM EDTA and 20 mM ammonium acetate, pH 4.4. The tocopherol (or tocopheryl quinone) was measured using a HPLC equipped with a Coulochem II Electrochemical Detector, a ESA Model 580 Solvent Delivery Module, a Column Catecholamine HR-80 (C18, 3 mm, 8 cm), a Model 5011 Analytical Cell, a Model 5020 Guard Cell (Chelmsford, MA), a PE Nelson 900 Series Interface with PE Nelson Turbochrom V4 software (Perkin Elmer, San Jose, CA). The flow rate was adjusted to 1.5 ml/min with E1 = -600 mV (the first analytical cell potential), E2 = 400 mV (the second analytical cell potential and E3 = 300 mV (the conditioning cell potential). The response factors of tocopherols (or tocopheryl quinones) relative to tocol were determined in triplicate. The concentrations of alpha-tocopherol, gamma-tocopherol, rac-5,7-tocol, alpha-tocopheryl quinone, and gamma-tocopheryl quinone in the standard solutions were measured using a Spectronic 3000 Array Spectrometer (Milton Roy, Rochester, NY) and published extinction coefficients .
Synthesis and purification of alpha-tocopheryl quinone (or gamma-tocopheryl quinone)
FeCl3·6H2O (1360 mg) was dissolved in 30 ml ethanol, and 530 mg of alpha-tocopherol (or gamma-tocopherol) was dissolved in 40 ml ethanol. Both solutions were mixed and placed in water bath at 50°C. After 3 hr, a 35 ml aliquot of the reaction mixture was placed in a separatory funnel, mixed with 50 ml of ether, washed with 50 ml of water, then washed with 50 ml of 0.15 M EDTA to chelate Fe ions. After the ether layer was dried by evaporation in a rotary evaporator, the crude quinone oil was dissolved in 10 ml ethanol and stored in a freezer at -20°C. The crude sample of tocopheryl quinone was purified using a HPLC system equipped with a Dynamax PC UV-M Multiple Wavelength Detector (Rainin, Woburn, MA), a model 2350 HPLC Pump, a C18 Column (5 mm, 250 mm) (ISCO, Lincoln, NE).