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2
*
Department of Vitamins and Atherosclerosis, German Institute of Human Nutrition, and
Institute of Nutritional Science, University of Potsdam, D-14558 Bergholz-Rehbruecke, Germany
2To whom correspondence should be addressed. E-mail: flohe{at}mail.dife.de.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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- and 
-tocotrienol was investigated in HepG2 cells. Metabolites were identified by HPLC and gas chromatography/mass spectrometry. -Tocotrienol was degraded to -CEHC (carboxyethyl hydroxychroman), -CMBHC (carboxymethylbutyl hydroxychroman), -CMHenHC (carboxymethylhexenyl hydroxychroman), -CDMOenHC (carboxydimethyloctenyl hydroxychroman) and -CDMD(en)2HC (carboxydimethyldecadienyl hydroxychroman). -Tocotrienol yielded -CEHC, -CMBHC, -CMHenHC and -CDMOenHC, whereas -CDMD(en)2HC could not be detected. These findings demonstrate that the trienols are metabolized essentially like tocopherols, i.e., by 
-oxidation followed by ß-oxidation of the side chain. The failure to detect CMBHC with the original double bond in the side chain reveals that auxiliary enzymes are involved, as in the metabolism of unsaturated fatty acids. CMBHC were the most abundant metabolites obtained from the tocotrienols as well as from -tocopherol. Quantitatively, the tocotrienols were degraded to a larger extent than their counterparts with saturated side chains. The pronounced quantitative differences in the metabolism between individual tocopherols as well as between tocotrienols and tocopherols in vitro suggest a corresponding lack of equivalence in vivo.
KEY WORDS: • tocotrienol • tocopherol • ß-oxidation • carboxyethyl hydroxychroman • metabolism
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-tocopherol in corn and soybean oils, tocotrienols in cereal grains, bran, some nuts and palm oil, -tocopherol is preferentially retained in the plasma. Forms of vitamin E not preferentially used are thought to be eliminated via the bile (1
). Biliary transport mechanisms for vitamin E, however, are not known. An alternative route of elimination may be the urinary excretion of vitamin E degradation products [see (2
,3
) for reviews]. Tocopherols are metabolized by side-chain degradation and elimination of the final products, carboxyethyl hydroxychromans (CEHC),3
in the urine. The structure of CEHC implies that the side chain has been degraded by a ß-oxidation pathway, which requires an initial -hydroxylation step. The ß-oxidation pathway has been confirmed by the identification of 
-CEHC (4
), -CEHC (5
,6
) and -CEHC (7
), its precursors, the carboxymethylbutyl hydroxychroman (CMBHC), derived from -, - and -tocopherol (8
,9
) and the -tocopherol-derived -carboxymethylhexyl hydroxychroman (-CMHHC) (10
) (Fig. 1
). More upstream -tocopherol–derived intermediates were identified recently (11
). The -oxidation has been proven indirectly by the inhibition of CEHC production from -, - and -tocopherol by sesamin and ketoconazole (12
), which are inhibitors of cytochrome P450 enzymes type 3A (CYP3A), and by a stimulation of -CEHC production by rifampicin (10
), an inducer of CYP3A. However, human CYP4F2 and not CYP3A4 hydroxylated - and -tocopherol most efficiently (11
). Thus, the metabolism of tocopherols appears to be achieved by CYP4F2-dependent -oxidation followed by ß-oxidation.
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). The structure of the final products again implies a degradation by an initial -hydroxylation followed by ß-oxidation steps as described for tocopherols. Intermediates of tocotrienol metabolism, however, have not yet been identified. We therefore investigated whether CEHC precursors are produced from - and -tocotrienol in HepG2 cells. Nearly all possible ß-oxidation intermediates were found from - and -tocotrienol, indicating that tocotrienols are indeed metabolized via ß-oxidation, as are tocopherols. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Acetic acid, tert-butyl methyl ether (TBME), ascorbic acid and tocopherols were obtained from Merck (Darmstadt, Germany). n-Hexane and acetonitrile (ACN) were purchased from Roth (Karlsruhe, Germany). Tetraethyl ammonium hydroxide (TEAH, 20% in water), BHT, EDTA as well as all chemicals for the synthesis of the internal standard were from Sigma (Deisenhofen, Germany). The -CEHC standard was synthesized by LABORAT GmbH (Berlin, Germany) (6
). R-- and R--tocotrienol were kindly provided by P. Hoppe, BASF AG, Ludwigshafen, Germany; -CMBHC by D.P.R. Muller, University College London, UK; and -CEHC (LLU-) by W. Wechter, Loma Linda University (Loma Linda, CA).
Cell culture.
Human hepatoma HepG2 cells (ATCC HB 8065) were cultured in 6-well plates at a density of 250,000 cells/well in 8 mL RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) containing <10 nmol/L -tocopherol, 2 mmol/L alanyl-glutamine, 100,000 U/L penicillamine and 100 mg/L streptomycin (Gibco, Eggenstein, Germany). Vitamin E–containing FCS was prepared by adding the corresponding vitamers from an ethanolic stock solution to the serum. The serum was left at 4°C overnight before being added to the medium. For the determination of proteins, cells were rinsed with PBS, scraped from the plate, centrifuged at 150 x g for 8 min and lysed by sonification in 1.5 mL PBS. Protein was determined with Coomassie reagent (Biorad, Munich, Germany) (14
).
Synthesis of -carboxypropyl hydroxychroman (-CPHC) as internal standard.
In principle, the synthesis was carried out according to the procedures described for the synthesis of -CMBHC by Pope et al. (15
), modified from (16
). The key intermediate, 5-hydroxy-5-methyl-hept-6-enoic acid ethyl ester, was obtained by addition of vinyl magnesium bromide (40 mL of a 1 mol/L solution in tetrahydrofuran) to acetyl butyric acid ethyl ester (40 mmol in 100 mL diethyl ether) under vigorous stirring (60 min, 0–5°C, argon atmosphere). The reaction mixture was stirred for additional 30 min, acidified with 1 mol/L HCl to pH 2.0 and diluted with water to dissolve precipitated salts. This solution was extracted with ether (3X 100 mL) and the combined extracts washed with a saturated sodium chloride solution (3X 50 mL) and dried over sodium sulfate. The ether was removed by evaporation. The resulting yellow oil was purified by silica gel chromatography (hexane/ethyl acetate, 8:1) to yield a transparent oil (4.5 g, 60%). The resulting 5-hydroxy-5-methyl-hept-6-enoic acid ethyl ester (1.9 g, 10.2 mmol) in 4 mL dioxane was added over 1.5 h at 110°C to a stirring solution of 2,3,5-trimethylhydroquinone (1.03 g, 6.8 mmol) and borone trifluoride diethyl etherate (1.72 mL, 13.6 mmol) in 20 mL dioxane under argon. The reaction mixture was extracted with ethyl acetate (3X 100 mL). The combined organic phases were washed with water, dried over Na2SO4, concentrated under vacuum and applied to silica gel chromatography (hexane/ethyl acetate, 4:3). The product was crystallized in ether/hexane at -20°C to yield a colorless solid (853 mg, 39%). Identity was confirmed by the mass spectra of the trimethylsilyl ether esters recorded on a gas chromatography/mass spectrometry (GC/MS) as described below, and by nuclear magnetic resonance (NMR). 1H and 13C NMR spectra were recorded on a Bruker AMX-300 spectrometer (Rheinstetten, Germany). Chemical shifts are reported as ppm relative to trimethylsilane (TMS) as internal standard. 1H NMR (CDCl3) 1.26 (s, 3H), 1.55–1.65 (m, 2H), 1.80 (m, 4H), 2.12 (s, 6H), 2.17 (s, 3H), 2.39 (m, 2H), 2.63 (t J=6.9 Hz, 2H). 13C NMR (CDCl3): 11.2 (5aC), 11.8 (8bC), 12.2 (7aC), 19.0 (4C), 20.6 (2'C), 23.6 (2aC), 31.5 (1'C), 34.2 (3C), 38.9 (3'C), 74.1 (2C), 117.2 (8C), 118.5 (5C), 121.1 (4aC), 122.6 (7C), 144.7 (8aC), 145.3 (6C), 179.0 (COOH). MS (EI) m/z of trimethylsilyl ether ester: 436 (100%, M+), 236 (25%), 73 (50%).
Analysis of vitamin E metabolites by HPLC.
After incubation, the cell culture medium was removed for sample preparation as follows: 100 µL ascorbate (0.23 mol/L water), 20 µL -CPHC (30 µmol/L in ethanol) and 1.6 mL sodium acetate buffer (0.1 mol/L, pH 4.5) were added to 8 mL cell culture medium. After vortexing, the sample was extracted 3 times with 10 mL TBME/BHT (TBME containing 1 mL/L of a 10 g BHT/L ethanol solution). The combined organic phases were evaporated to dryness and redissolved in 200 µL of HPLC loading buffer (50 mL acetonitrile, 50 mL HPLC buffer, 440.8 mg BHT).
Metabolites were separated in a Summit HPLC-system with an ED 50 electrochemical detector (Dionex, Idstein, Germany) and a 250 x 4 mm, RP-18 end-capped column (Merck, Darmstadt, Germany) with a preceding guard column (4 x 4 mm) containing the same stationary phase. HPLC buffer consisted of TEAH (20% in water)/water/acetonitrile (25:540:430) with 0.63 mmol/L EDTA. The pH was adjusted to 5.5 with acetic acid. The flow was set to 0.6 mL/min with a gradient program of 0% ACN: 0–34 min; 0–45% ACN: 34–52 min; 45–96% ACN: 52–55 min; 96% ACN: 55–65 min; 96–0% ACN: 65–68 min, and 0% ACN: 68–78 min. For coloumetric detection, the analytical cell was set to +0.55 V for all metabolites.
Estimation of response factors.
Varying concentrations of available authentic metabolites (-CEHC, -CMBHC, and -CEHC) and a constant concentration of the internal standard -CPHC were added to fresh cell culture medium. Metabolites were extracted as described above, separated by HPLC and quantified by means of -CPHC. The response factors were calculated as the ratio of metabolite concentration applied to the metabolite concentration calculated via the internal standard.
Identification of tocotrienol metabolites by GC/MS.
Compounds eluting from the HPLC-column were collected by a time-programmed fraction collector from Gilson Abimed (Langenfeld, Germany), reextracted as described above and derivatized in hexane as described (6
). Trimethylsilyl ether esters were analyzed by GC/MS with an SSQ 710 MAT from Finnigan MAT (Bremen, Germany) with a Varian (Santa Clarita, CA) gas chromatograph (6
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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HepG2 cells incubated with 50 µmol/L - or -tocotrienol, respectively, released tocotrienol metabolites, which were separated by HPLC. -Tocotrienol metabolites (Fig. 2A
) appeared at 9.53 (peak 1a), 30.19 (peak 2a) and 49.08 min (peak 3a). The lipophilic material eluting during the wash phase (60–75 min; Ea) was not adequately separated. Therefore, fraction Ea was collected and analyzed by GC/MS, using for detection the mass fragment at m/z 237, which is indicative of an -substituted chroman structure (see below). As shown in the insert of Figure 2
A, a single peak fulfilling this criterion was detected (4a). Accordingly, -tocotrienol metabolites (Fig. 2
B) eluted at 7.45 (1b), 20.48 (2b) and 34.19 min (3b) and a poorly dissolved fraction Eb in the wash phase (60–75 min). GC/MS analysis of Eb by means of the mass fragment characteristic of the -substituted chroman ring (m/z 223) yielded two potential metabolites, 4b and 5b (insert to Fig. 2
B). All peaks were collected and analyzed by GC/MS.
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HPLC peaks 1a,b and 2a,b corresponded to -, -CEHC and -, -CMBHC, respectively, as confirmed by authentic standards in HPLC and GC/MS analyses (not shown). The mass spectrum of the as yet unknown peak 3a revealed the fragment at m/z 237, which is typical for the fragmented -substituted chroman ring, the ion at m/z 73 derived from the TMS group and a molecular ion at m/z 490 (Fig. 3A
). The mass of 490 corresponds to the derivatized precursor of -CMBHC, 2,5,7,8-tetramethyl-2-(6'-carboxy-4'-methylhex-4'-enyl)-6-hydroxychroman (-CMHenHC). The mass spectrum of peak 4a showed the mass of m/z 237 for which it was preselected, the TMS fragment (m/z 73), and the molecular ion with a mass of m/z 532 (Fig. 3
B). The latter complies with 2,5,7,8-tetramethyl-2-(8'-carboxy-4',8'-dimethyloct-4'-enyl)-6-hydroxychroman (-CDMOenHC).
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-tocotrienol metabolites compiled in Figure 4
all displayed the mass fragment m/z 223 characteristic of the -substituted chroman ring and the TMS fragment (m/z 73). The molecular masses corresponded to derivatives of three distinct metabolites that differ in the progression of ß-oxidation: 3b (m/z 476) to 2,7,8-trimethyl-2-(6'-carboxy-4'-methylhex-4'-enyl)-6-hydroxychroman (-CMHenHC) (Fig. 4
A); 4b (m/z 518) to 2,7,8-trimethyl-2-(8'-carboxy-4',8'-dimethyloct-4'-enyl)-6-hydroxychroman (-CDMOenHC) (Fig. 4
B); and 5b (m/z 544) to 2,7,8-trimethyl-2-(10'-carboxy-4',8'-dim-ethyldeca-4',8'-dienyl)-6-hydroxychroman [-CDMD(en)2HC]) (Fig. 4
C). The amount of the analogous metabolite of -tocotrienol was too low to be identified.
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For the quantification of vitamin E metabolites with electrochemical detection (ECD), a reliable internal standard was required. The standard should have a structure similar to the vitamin E metabolites and should react identically in the ECD. Therefore, an artificial intermediate of the ß-oxidation pathway of tocols with 4 C-atoms in the side chain, -CPHC, was synthesized. In the HPLC chromatogram, -CPHC exceeded 99.9% purity. It eluted between CEHC and CMBHC. The response factors determined for the available authentic metabolites in relation to -CPHC were as follows: -CEHC, 0.925 ± 0.083; -CMBHC, 0.756 ± 0.055; and -CEHC, 0.964 ± 0.066 (n = 3 each). Response factors for CEHC were close to 1.0, indicating the almost identical behavior of the internal standard and these metabolites in the sample preparation procedure and the detection mode. Under the assumption that - and -CMBHC had response factors similar to - and -CEHC, -CMBHC was quantified using the response factor of -CMBHC.
From the naturally occurring isoform RRR--tocopherol as well as the synthetic all-rac--tocopherol, only trace amounts of -CEHC were released from HepG2 cells (Fig. 5
), corroborating previous experiments that showed that -CEHC was either not released at all (9
) or only after a long incubation time (10
). With concentrations of 0.061 and 0.052 nmol -CMBHC/mg protein from RRR- and all-rac--tocopherol, respectively, the amounts of -CMBHC exceeded that of -CEHC by a factor of 2–4. Incubation with -tocopherol resulted in the release of equal amounts of -CEHC and -CMBHC (1.2 nmol/mg protein). Although only small amounts of -CEHC were found from -tocotrienol (0.2 nmol/mg protein), substantial amounts of -CEHC were produced from -tocotrienol (2.2 nmol/mg protein). The release of CMBHC exceeded that of CEHC by a factor of 22 in the case of -tocotrienol and of 2.2 in the case of -tocotrienol. With the exception of -tocopherol, all types of vitamin E tested yielded higher amounts of CMBHC than of CEHC.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The metabolism of both tocopherols and tocotrienols results in CEHC as final products. We showed here that tocotrienols and tocopherols are degraded by essentially the same mechanism, an initial -hydroxylation followed by five cycles of ß-oxidation. In case of -tocotrienol, HepG2 cells released each of the possible carboxylic acid intermediates of the side-chain degradation (Fig. 6
). With -tocotrienol, three of the four possible -CEHC precursors were detected. The identification of - and -CMBHC, instead of a metabolite still containing the double bond present in the precursors, - and -CMHenHC, reveals that the metabolism of the tocotrienols is more complex than that of the tocopherols. As in the ß-oxidation of unsaturated fatty acids, auxiliary enzymes must be involved in tocotrienol metabolism. Formation of the ,ß-unsaturated fatty acid in the second and fourth cycles of the ß-oxidation of the tocotrienols leads to compounds with two conjugated double bonds, which are not accepted by enoyl-CoA hydratase that usually forms the ß-hydroxyacyl-CoA. In linoleic acid metabolism, for instance, this impasse is resolved by the action of two auxiliary enzymes, 2,4-dienoyl-CoA reductase and 3,2-enoyl-CoA isomerase (see textbooks of biochemistry). The identification of -CDMOenHC and -CMBHC thus reveals that the unsaturated side chain of tocotrienols is indeed metabolized like unsaturated fatty acids. Whether the initial -oxidation is catalyzed by identical or different hydroxylating enzymes remains to be investigated.
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In HepG2 cells, the different forms of vitamin E yielded different amounts of metabolites. Only low levels of -CEHC were obtained from -tocopherol, whether natural or synthetic. Concentrations of -CMBHC were generally higher but still small compared with -tocotrienol–derived -CMBHC. Surprisingly, only traces of -CEHC were recovered from -tocotrienol–loaded cells, whereas substantial amounts of -CEHC were derived from -tocopherol and -tocotrienol. Low levels of -tocopherol metabolites compared with those from -tocopherol were observed previously in cultured cells (9
) as well as in vivo (17
). On the basis of CEHC, only 1–3% of ingested RRR--tocopherol irrespective of the dosage applied was degraded to urinary metabolites in humans (8
). The portion of -CEHC derived from all-rac--tocopherol was shown to be 2–4 times higher (18
). In contrast, up to 50% of ingested -tocopherol was suggested to be degraded to -CEHC (17
).
To some extent, the tocopherol binding or transfer proteins, which display a pronounced preference for -tocopherol, may account for the metabolic differences by efficiently competing with the metabolizing system. In liver, the -tocopherol transfer protein (-TTP) specifically sorts out -tocopherol from all incoming tocopherols for incorporation into VLDL and transfer into the plasma (19
). -TTP binds RRR--tocopherol with the highest affinity, whereas those of SRR--tocopherol, -tocopherol and -tocotrienol are only 11, 9 and 12%, respectively (20
). This explains the low plasma levels of tocopherols other than -tocopherol. Also the cytosolic -tocopherol–associated protein (TAP) preferentially binds -tocopherol (21
,22
). In this way, -TTP and TAP may specifically protect -tocopherol from degradation and guide it to cellular sites where it can exert its specific regulatory roles (3
,23
).
The metabolic fate of tocotrienols is less well understood. In our cell culture model, concentrations of tocotrienol-derived CMBHC were generally higher than those of CEHC. Due to the lack of authentic standards, the absolute concentrations of CMBHC-precursors could not be determined. However, the quantification by ECD makes use of redox properties common to all intermediates, i.e., the hydroxy group at position 6 of the chroman ring. In addition, the response factors of CEHC and CMBHC with CPHC as internal standard were similar. It can be assumed, therefore, that peak areas obtained by HPLC provide a reasonable estimate also for the concentrations of the more upstream intermediates. With these precautions, CMBHC are the main products of tocotrienol metabolism in the cellular system. This observation suggests that the CMBHC/CEHC conversion is slower than any of the preceding ß-oxidation steps.
In vivo, plasma tocotrienol levels cannot be substantially increased even when the intake is high (24
); 2 and 6% of - and -tocotrienol have been found as - and -CEHC in human urine, respectively (13
). In view of the high amounts of CMBHC produced from tocotrienols by HepG2 cells, their occurrence could also be expected in vivo. In urine, however, CMBHC, although detectable, are found in consistently lower amounts than CEHC (8
,9
). This is not surprising because their higher lipophilicity makes their preferred route of excretion be via the bile rather than via the urine. Certainly, a more complete balancing of vitamin E metabolites comprising biliary or at least fecal excretion products is required to understand the pronounced effect of substituents at the chroman ring and the (stereo)chemistry of the side chain on the metabolic pattern.
In conclusion, we have shown that tocopherols and tocotrienols are degraded essentially by the same metabolic pathway. However, the extent of metabolization differs greatly in vitro. If this also holds true for the in vivo situation, the metabolic rate of individual forms of vitamin E should markedly affect their bioavailability and bioequivalence.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: ACN, acetonitrile; -TTP, -tocopherol transfer protein; CDMD(en)2HC, carboxydimethyldecadienyl hydroxychroman; CDMOenHC, carboxydimethyloctenyl hydroxychroman; CEHC, carboxyethyl hydroxychroman; CMBHC, carboxymethylbutyl hydroxychroman; CMHenHC, carboxymethylhexenyl hydroxychroman; CMHHC, carboxymethylhexyl hydroxychroman; CPHC, carboxypropyl hydroxychroman; CYP, cytochrome P450; ECD, electrochemical detection; FCS, fetal calf serum; GC/MS, gas chromatography/mass spectrometry; NMR, nuclear magnetic resonance; TAP, -tocopherol–associated protein; TBME, tert-butyl methyl ether; TEAH, tetraethyl ammonium hydroxide; TOH, tocopherol; TMS, trimethylsilane.
Manuscript received 7 June 2002. Initial review completed 3 July 2002. Revision accepted 26 July 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Kayden, H. J. & Traber, M. G. (1993) Absorption, lipoprotein transport, and regulation of plasma concentrations of vitamin E in humans. J. Lipid Res. 34:343-358.[Medline]
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18. Traber, M. G., Elsner, A. & Brigelius-Flohé, R. (1998) Synthetic as compared with natural vitamin E is preferentially excreted as -CEHC in human urine: studies using deuterated -tocopheryl acetates. FEBS Lett 437:145-148.[Medline]
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 J. Neuzil, M. Tomasetti, Y. Zhao, L.-F. Dong, M. Birringer, X.-F. Wang, P. Low, K. Wu, B. A. Salvatore, and S. J. Ralph Vitamin E Analogs, a Novel Group of "Mitocans," as Anticancer Agents: The Importance of Being Redox-Silent Mol. Pharmacol., May 1, 2007; 71(5): 1185 - 1199. [Abstract] [Full Text] [PDF]
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 K. S. Ahn, G. Sethi, K. Krishnan, and B. B. Aggarwal {gamma}-Tocotrienol Inhibits Nuclear Factor-{kappa}B Signaling Pathway through Inhibition of Receptor-interacting Protein and TAK1 Leading to Suppression of Antiapoptotic Gene Products and Potentiation of Apoptosis J. Biol. Chem., January 5, 2007; 282(1): 809 - 820. [Abstract] [Full Text] [PDF]
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B.-L. Song and R. A. DeBose-Boyd Insig-dependent Ubiquitination and Degradation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Stimulated by {delta}- and {gamma}-Tocotrienols J. Biol. Chem., September 1, 2006; 281(35): 25054 - 25061. [Abstract] [Full Text] [PDF]
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 R. S Bruno, S. W Leonard, J. Li, T. M Bray, and M. G Traber Lower plasma {alpha}-carboxyethyl-hydroxychroman after deuterium-labeled {alpha}-tocopherol supplementation suggests decreased vitamin E metabolism in smokers Am. J. Clinical Nutrition, May 1, 2005; 81(5): 1052 - 1059. [Abstract] [Full Text] [PDF]
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S. W. Leonard, E. Gumpricht, M. W. Devereaux, R. J. Sokol, and M. G. Traber Quantitation of rat liver vitamin E metabolites by LC-MS during high-dose vitamin E administration J. Lipid Res., May 1, 2005; 46(5): 1068 - 1075. [Abstract] [Full Text] [PDF]
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R. S. Bruno and M. G. Traber Cigarette Smoke Alters Human Vitamin E Requirements J. Nutr., April 1, 2005; 135(4): 671 - 674. [Abstract] [Full Text] [PDF]
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S. Schaffer, W. E. Muller, and G. P. Eckert Tocotrienols: Constitutional Effects in Aging and Disease J. Nutr., February 1, 2005; 135(2): 151 - 154. [Abstract] [Full Text] [PDF]
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C.-S. You, T. J. Sontag, J. E. Swanson, and R. S. Parker Long-Chain Carboxychromanols Are the Major Metabolites of Tocopherols and Tocotrienols in A549 Lung Epithelial Cells but Not HepG2 Cells J. Nutr., February 1, 2005; 135(2): 227 - 232. [Abstract] [Full Text] [PDF]
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