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.

Cell culture

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 [15].

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).

The uptake and depletion of tocopherols (separately and together)

Confluent RAW264.7 macrophages were incubated with either 5.6 μM alpha-tocopherol or 4.9 μM gamma-tocopherol (Figure 3A) or with both tocopherols together (Figure 3B with alpha-tocopherol at 4.6 μM and gamma-tocopherol at 5.2 μM) and the uptake of tocopherols measured during 6 hours of incubation. Uptake is defined as the net difference between tocopherol transported into the cells and loss due to catabolism and/or in vitro oxidation. After 6 hours, the medium with tocopherols was removed and replaced with medium not containing tocopherols and depletion of tocopherols followed for 4 hours.

After 6 hours of incubating macrophages with either alpha- or gamma-tocopherol (Figure 3A), the intracellular levels of gamma-tocopherol were at least six fold higher than the levels of alpha-tocopherol (p < 0.001). A major goal of this research was to determine if alpha-tocopherol and gamma-tocopherol interacted with each other so as influence their uptake and depletion. Figure 3B shows the uptake and depletion of alpha-tocopherol and gamma-tocopherol when both were incubated together with macrophages. It is clear (compare Figure 3A to 3B) that the presence of gamma-tocopherol considerably enhanced the uptake of alpha-tocopherol (p < 0.001). The presence of alpha-tocopherol did not, however, markedly enhance the uptake of gamma-tocopherol. After washing with fresh medium without tocopherols, the macrophage levels of both alpha-tocopherol and gamma-tocopherol decreased, but the depletion of gamma-tocopherol was always faster than that of alpha-tocopherol.

It is interesting that the depletion of alpha-tocopherol from the medium during the uptake phase was always greater than that of gamma-tocopherol yet the cellular uptake of gamma-tocopherol was always greater than that of alpha-tocopherol. Tables 1 and 2 also indicate that gamma-tocopherol decreases from RAW264.7 macrophages more rapidly than alpha-tocopherol during the depletion phase.

If tocopheryl quinones were the only catabolic/oxidation products formed during incubation with the macrophages, then the nmoles of tocopherol plus the nmoles of tocopheryl quinone in the medium and in the cells should be constant. Table 3 clearly shows that the sum of the tocopherol and its quinone product decreased over the 6 hr experiment suggesting that products, other than the tocopheryl quinone, were formed during the experiment. Moreover, these data show that after 6 hr of incubation: (1) cellular alpha-tocopheryl quinone levels were generally higher than those of gamma-tocopheryl quinone; (2) cellular tocopheryl quinones levels were always much lower than cellular tocopherol levels. This latter point suggests that the low cellular levels of alpha-tocopherol (compared to gamma-tocopherol levels) shown in Figure 3A, as compared to Figure 3B, cannot be explained by the simple cellular oxidation of alpha-tocopherol to alpha-tocopheryl quinone (with no further catabolism or oxidation). After 6 hr, macrophages incubated with both alpha- and gamma-tocopherol accumulated about 0.5 nmole more alpha-tocopherol than did macrophages incubated with alpha-tocopherol alone. If, in the absence of gamma-tocopherol, 0.5 nmole of alpha-tocopherol were converted to alpha-tocopheryl quinone (and no further catabolism occurred) than this should be recovered in cells. The data in Table 3 indicate, however, a cellular accumulation of only about 0.03 nmoles of alpha-tocopheryl quinone. Moreover, macrophages actually had a lower content of alpha-tocopheryl quinone when incubated with alpha-tocopherol alone then when incubated with both alpha- and gamma-tocopherol together. Although gamma-tocopherol promotes the cellular uptake of alpha-tocopherol, it clearly did not do so by dramatically protecting alpha-tocopherol from oxidation to alpha-tocopheryl quinone (with no further catabolism/oxidation of alpha-tocopheryl quinone).

The uptake and depletion tocopheryl quinones (separately and together)

RAW 264.7 macrophages were incubated with 0.6 μM alpha-tocopheryl quinone or 0.4 μM gamma-tocopheryl quinone alone (Figure 4A) or with both together (Figure 4B with 0.6 μM alpha-tocopheryl quinone and 0.4 μM gamma-tocopheryl quinone) and the uptake and depletion measured as described above. Figure 4 indicates that alpha-tocopheryl quinone accumulates to higher levels than gamma-tocopheryl quinone (e.g. at 6 hours the difference is significant at p < 0.001). Similarly, alpha-tocopheryl quinone was lost from the cells more rapidly during the depletion phase. These results are consistent with those in Table 3, which always indicate a higher intracellular concentration of alpha-tocopheryl quinone compared to gamma-tocopheryl quinone.

The uptake and depletion of tocopherols (separately and together)

Our results consistently show that gamma-tocopherol was taken up more rapidly and accumulated to higher intracellular levels than alpha-tocopherol. Moreover, the presence of gamma-tocopherol markedly promoted the uptake of alpha-tocopherol by macrophages. When macrophages were incubated with alpha-tocopherol or gamma-tocopherol individually, the macrophage level of alpha-tocopherol was just 10–20% of that of gamma-tocopherol (Figure 3A). In marked contrast, when macrophages were incubated with both alpha- and gamma-tocopherols together the cellular level of alpha-tocopherol were 70–80% (after 6 hr) that of gamma-tocopherol (Figure 3B). There were no marked changes in the levels of gamma-tocopherol caused by the presence of alpha-tocopherol.

Tocopherol quinones were considered to be likely oxidization products of the tocopherols. The increased cellular uptake of alpha-tocopherol caused by gamma-tocopherol could be caused by gamma-tocopherol protecting alpha-tocopherol from in vitro oxidation to its quinone form. However, the data in Table 3 show that gamma-tocopherol does not slow the oxidation of alpha-tocopherol to alpha-tocopheryl quinone. In the presence of gamma-tocopherol, the cellular levels of alpha-tocopheryl quinone were even higher than in its absence (Table 3). It is possible, however, that gamma-tocopherol may protect alpha-tocopherol from catabolism (or in vitro oxidation) into products other than quinones. The data in Table 3 certainly suggest that products, other than quinones, were produced when the macrophages were incubated with tocopherols. Those metabolites could very possibly be water-soluble products, such as alpha-CEHC or gamma-CEHC that were not measured in this investigation.

It is not clear why our result differ from those reported by Jiang et al. [14] who also measured the uptake of alpha- and gamma-tocopherol separately in RAW264.7 macrophages (but not both together). Since gamma-tocopherol is depleted from macrophages more rapidly than alpha-tocopherol the time interval between removal of the tocopherol enriched medium and cell harvesting could be an important variable. Our results are, however, qualitatively very similar to those reported by Tran and Chen [13] for human endothelial cells although these investigators did not directly compare the results from incubating cells with alpha-tocopherol and gamma-tocopherol separately or together.

The uptake and depletion tocopheryl quinones (separately and together)

When macrophages were incubated tocopheryl quinones (see Figure 4), the cellular levels of alpha-tocopheryl quinone were always found to be higher than that of gamma-tocopheryl quinone (independent of the presence of gamma-tocopheryl quinone). The data in Table 4 suggest that alpha-tocopheryl quinone is fairly stable in the cell incubation system compared to gamma-tocopheryl quinone.

Physiological significance of the results

It is not known if the in vitro results of this study are relevant to the in vivo conditions. It is interesting, however, that in an animal model, graded levels of dietary gamma-tocopherol in a diet containing a constant level of alpha-tocopherol were found to increase the levels of alpha-tocopherol in serum and most tissues [16]. This remarkable result has yet to be reproduced in humans but certainly suggests an interaction between dietary gamma-tocopherol and alpha-tocopherol similar to that reported here on the cellular level. The potential health related significance of gamma-tocopherol is being increasingly recognized [17].