p-Oxidation of tocopherol and tocotrienol depends on initial m-hydroxylation and subsequent oxidation of the hydroxyl group to an aldehyde and carboxylic acid function. The involvement of cytochrome P450 (CYP) enzymes appears plausible and has been deduced from inhibition and induction studies. Ketoconazole and sesamin, then believed to affect CYP3A-type enzymes specifically, have been shown to inhibit the release of y- and S-CEHC and y- and S-CMBHC from HepG2 cells (29). Rifampicin, known as a CYP3A inducer, stimulated the release of a-CEHC from HepG2 cells when treated with all-rac-a-tocopherol but not when treated with RRR-a-tocopherol (27). Recently, however, microsomes from insect cells transfected with human CYP4F2 have been shown to be most active in oxidizing the m-methyl group of RRR-a- and y-tocopherol, whereas a variety of other cytochromes including CYP3A4 were inactive (28). In this system, CYP4F2 preferentially degraded y-tocopherol, whereas a-tocopherol was bound slightly better but metabolized more slowly. This was interpreted to explain the comparatively slow turnover of RRR-a-tocopherol in general. Difficulties in detecting a-CEHC release from HepG2 cells might, however, equally result from other reasons. We have shown that RRR-a-tocopherol degrades only after a long incubation time or in cells already adapted to all-rac-a-tocopherol (27). The inability of rifampicin to up-regulate RRR-a-tocopherol degradation could point to the involvement of a different CYP in the metabolism of this stereoisomer. In addition, the experimental results shown in Figure 12.1 may be interpreted along these lines The yield of degradation products from HepG2 cells was substantially higher with y-tocopherol and could not be further increased by rifampicin treatment, whereas rifampicin stimulated all-rac-a-tocopherol-derived a-CEHC and a-CMBHC release. If not due to different CYP, the differential rates and inducibilities of the tocopherol metabolism may result from the following: (i) highly different affinities or metabolizing rates of the hydroxylation system for a- and y-tocopherol as suggested (28); (ii)
Fig. 12.1. Rifampicin stimulates the release of metabolites from all-rac-a-tocopherol but not from y-tocopherol from HepG2 cells into the culture medium. Tocopherol-adapted HepG2 cells (27) were deprived of a-tocopherol by incubation in tocopherol-free medium for 4 d (wash-out). After that, no metabolites were detectable in the cell culture medium. During the last 2 d of the wash-out period, 50 pmol/L rifampicin was added to the culture medium. Control cells did not receive rifampicin. After the complete washout phase, 100 pmol/L all-rac-a- or y-tocopherol was added. Afer 72 h, the medium was collected and 1-naphtol added as internal standard (final concentration 0.033 pmol/L). Thereafter, the medium was acidified to pH 4.5 and extracted 3 times with 10 mL t-butylmethylether. Solvent was removed by evaporation; the resulting residue was dissolved in high-performance liquid chromatography (HPLC) solvent and analyzed by HPLC-electrochemical detection as described (27). To obtain faster elution, acetonitrile was mixed with the solvent from min 32 to reach 40% acetonitrile after 52 min. Because an authentic standard was not available for y-carboxymethylbutyl hydroxychro-man (y-CMBHC), data are expressed as 1-naphtol equivalents (NE). Response factors were 1.46 and 1.48 for a- and y-carboxyethyl hydroxychromans (CEHC), respectively, and 2.44 for a-CMBHC. Actual concentrations (pmol/mg protein) were: 29.6 ± 16.2 for a-CEHC (control); 91.3 ± 42.5 for a-CEHC (rifampicin); 602 ± 176 for a-CMBHC (control); 1471.5 ± 397.5 for a-CMBHC (rifampicin). The concentrations of y-CEHC (nmol/mg protein) were: 3.89 ± 0.71 (control) and 4.08 ± 1.32 (rifampicin). Thus, concentrations of a-CMBHC are somewhat underestimated in the figure and probably also those of y-CMBHC. Values are means ± SD (n = 3). For better clarity, the a-CEHC is shown again in the insert on a different scale.
effective binding of a-tocopherol to a-TTP or to one of the tocopherol-associated proteins (TAP) (30,31), thereby preventing degradation; or (iii) upregulation of the metabolizing system by Y-tocopherol itself. Which of the possibilities finally turns out to be true remains to be determined.
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