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ω-Hydroxylation of α-tocopheryl quinone reveals a dual function for cytochrome P450-4F2 in vitamin E metabolism
Bioorganic & Medicinal Chemistry 26 (2018) 5555–5565
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ω-Hydroxylation of α-tocopheryl quinone reveals a dual function for cytochrome P450-4F2 in vitamin E metabolism
T
Luke Taylorb, Nick Kruegerb, Olga Malyshevaa, Jeffrey Atkinsonb, , Robert S. Parkera, ⁎
a b
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Division of Nutritional Sciences, Savage Hall, Cornell University, Ithaca, NY 14853, USA Department of Chemistry and Centre for Biotechnology, Brock University, 1812 Sir Isaac Brock Way, St. Catharines, Ontario L2S 3A1, Canada
ARTICLE INFO
ABSTRACT
This paper is dedicated to David Muller and Graham Burton, two giants of vitamin E biology.
α-Tocopherol (α-TOH) is the primary lipophilic radical trapping antioxidant in human tissues. Oxidative catabolism of α-tocopherol (αTOH) is initiated by ω-hydroxylation of the terminal carbon (C-13) of the isoprenoid sidechain followed by oxidative transformations that sequentially truncate the chain to yield the 2,5,7,8-tetramethyl(3′carboxyethyl)-6-hydroxychroman (α-CEHC). After conjugation to glucuronic acid, 3′-carboxyethyl-6hydroxychroman glucuronide is excreted in urine. We report here that the same enzyme that accomplishes this task, the cytochrome P450 monooxygenase CYP-4F2, can also ω-hydroxylate the terminal carbon of α-tocopheryl quinone. A standard sample of ω-OH-α-tocopheryl quinone (ω-OH-α-TQ) was synthesized as a mixture of stereoisomers by allylic oxidation of α-tocotrienol using SeO2 followed by double-bond reduction and oxidation to the quinone. After incubating human liver microsomes or insect cell microsomes expressing only recombinant human CYP-4F2, cytochrome b5, and NADPH P450 reductase with d6-α-tocopheryl quinone (d6-αTQ), we showed that the ω-hydroxylated (13-OH) d6-α-TQ was produced. We further identified the production of the terminal carboxylic acid d6-13-COOH-αTQ. The ramifications of this discovery to the understanding of tocopherol utilization and metabolism, including the quantitative importance of the αTQ-ω-hydroxylase pathway in humans, are discussed.
Keywords: Vitamin E α-Tocopherol α-Tocopheryl quinone Cytochrome P450-4F2 ω-Hydroxylation Oxidative metabolism
1. Introduction α-Tocopherol (αTOH) is the primary lipophilic antioxidant in cell membranes, lipoproteins, and lipid droplets. It functions by donating electrons to highly reactive lipid peroxides, thereby reducing them to less reactive lipid hydroperoxides. One mole of αTOH is capable of reducing two moles of lipid peroxide, first by using the phenolic hydrogen on the chromanol ring, forming the chromanoxyl radical of αTOH (αTO•). αTO• is a relatively stable radical, with a half-life of approximately 5 min in hexadecyltrimethylammonium chloride and milliseconds in DPPC bilayers (1, 2), and has two fates: (a) reduction to αTOH by small reducing substances such as reduced ubiquinone, ascorbic acid, and glutathione (see1 and references therein), or (b) reaction with a second lipid peroxyl radical (although see2 for alternative chemistries). This second reaction ultimately results in the irreversible oxidation of αTO• a to α-tocopheryl quinone (αTQ), as illustrated in Fig. 1 below. The metabolic fate of αTQ is at present unknown and is the subject of this investigation. Cytochrome P450-4F2 (CYP4F2) is an NADPH-dependent endoplasmic reticulum enzyme whose primary function appears to be the
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initiation of the oxidative degradation and elimination of tocopherols and tocotrienols3, thereby preventing their over-accumulation in cell membranes. CYP4F2 catalyzes the hydroxylation of one of the two terminal methyl groups on the tocochromanols, yielding (in the case of αTOH) ω-OH-αTOH. ω-OH-αTOH is then oxidized by a microsomal dehydrogenase to ω-COOH-αTOH. This intermediate then undergoes a series of oxidative chain-shortening reactions analogous to that of branched-chain fatty acids, ultimately yielding 2,5,7,8-tetramethyl (3′carboxyethyl)-6-hydroxychroman, or α-CEHC, which is glucuronidated and excreted in the urine, as illustrated in Fig. 11 in the Discussion section below. To date, CYP4F2 is the only enzyme proven to exhibit vitamin E-ω-hydroxylase activity. Sontag and Parker3 reported on the results of testing all available CYP enzymes (commercially available or gifts), including CYP3A4, for this activity. Only CYP4F2 tested positive. We hypothesized that αTQ undergoes a pathway of oxidative catabolism similar to that of αTOH, initiated by microsomal CYP4F2. In order to demonstrate this in the most rigorous fashion, we used deuterium-labeled αTQ as substrate and chemically synthesized the putative CYP4F2 reaction product, ω-OH-αTQ for use as a standard to probe
Corresponding authors. E-mail addresses: [email protected] (J. Atkinson), [email protected] (R.S. Parker).
https://doi.org/10.1016/j.bmc.2018.10.002 Received 24 August 2018; Received in revised form 28 September 2018; Accepted 4 October 2018 Available online 05 October 2018 0968-0896/ © 2018 Elsevier Ltd. All rights reserved.
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Fig. 1. Conversion of αTOH to αTQ by two-electron oxidation. After primary oxidation, αTO• may combine with a second peroxyl radical which is then hydrolyzed to yield αTQ. Alternatively, metal assisted single electron oxidation proceeds through the tocopherone cation2.
Scheme 1. Terminal allylic hydroxylation of α-tocotrienol acetate. (See Table 2 in Supplementary Information for variations in conditions and the corresponding isolated yields.).
formation of the deuterated metabolite by human liver microsomes and insect cell microsomes containing recombinant CYP4F2 and its reaction partners NADPH-CYP-reductase and cytochrome-b5.
microsomes (HLM) were purchased from Corning Incorporated (Corning NY). NADPH and NAD were purchased from Sigma-Aldrich Co. (St. Louis, MO). The microsomal reaction substrate d6-α-tocopherylquinone (d6αTQ) was synthesized in our lab via the FeCl3 oxidation of d6-α-TOH (a gift from Dr. Graham Burton) and used the in microsomal system as a bovine serum albumin (BSA) complex as described previously6.
2. Methods 2.1. Synthesis of d0-ω-hydroxy-α-tocopheryl quinone Having access to neither a convenient supply of garcinoic acid (from Garcinia kola)4 nor the desire to complete a multistep total synthesis5, we decided that it might be convenient to attempt a synthesis of a terminally hydroxylated α-tocopherol and its quinone by a selective terminal allylic oxidation of α-tocotrienol of which we had a large supply in hand. Catalytic reduction of the double bonds, reductive removal of the phenol protecting group and oxidation to the quinone would provide the desired ω-OH-α-tocopherylquinone (Scheme 1) Full synthetic and analytical data are provided in the Supplementary Material.
2.3. Microsomal reaction system Appropriate volumes of 4.4 mM d6-αTQ BSA complex were added to microsome suspensions consisting of either HLM (20 mg/ml protein) or CYP4F2 insect microsomes (100 pmol CYP/mL) and a sufficient volume of 100 mM KH2PO4 buffer pH 7.2, total volume 0.475 ml. This system was preincubated for 10 min in open glass culture tubes in a 37 °C water bath. NADPH and NAD (if used), final reaction concentrations of 1 mM, were added for a final reaction volume of 0.5 ml. Samples were incubated at 37 °C for an additional 20 min then immediately chilled on ice. d0-ω-Hydroxy-αTQ (the internal standard, in 5 μL ethanol) was then added and the microsomal system extracted first with 2 ml 95% ethyl acetate and then with 2 ml 1:1 hexanes:MTBE. The extracts were combined, evaporated to dryness under a stream of N2 and the residue dissolved in 100 μL acetonitrile for LCMS analysis, or in silylation reagents for GC-MS analysis.
2.2. Microsomes and deuterated αTQ substrate Recombinant insect cell microsomes expressing recombinant human CYP4F2, cytochrome b5, and NADPH P450 reductase, control insect cell microsomes devoid of all CYP enzymes, and pooled human liver
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A
B
Fig. 2. A: LC mass spectrum of d6-αTQ. B: Fragmentation of d6-αTQ, yielding the m/z 171 fragment ion.
2.4. LC-MS and GC-MS analysis of microsomal reaction products
flow; from 30 min to 35 min, the mobile phase consisted of a linear gradient changing from 99% to 80% B; finally, the column was equilibrated to initial conditions for another 5 min with 80%B. Flow rate was 0.3 ml/min. The Q Exactive mass spectrometer was equipped with a HESI probe and operated in positive ionization mode with parameters of: heater temperature, 30 °C; sheath gas, 40 (arbitrary units), auxiliary gas, 10 ml/min; sweep gas, 3 ml/min; spray voltage, 5 kV. Capillary temperature was set at 350 °C, and S-lens was 50. A full scan range was set at 50–670 (m/z). The resolution was set at 35,000. The maximum injection time (max IT) was 200 ms. Automated gain control (AGC) was targeted at 3 × 106 ions. For All Ion Fragmentation (AIF) analysis normalized energy collision energy (NCE) was 25%. Quantification of metabolites was performed using
UPLC/MS system Ultimate 3000 UHPLC (Dionex) coupled to Q Exactive-Mass spectrometer (QE-MS, Thermo Scientific, San Jose, CA) was used for chromatographic separation and peak analysis. Separation of d6 -a-tocopherol and metabolites was achieved by reversed phase liquid chromatography using an Acclaim RSLC 120 C18 column (2.1 × 150mm, 2.2 μm, Thermo Scientific) and multistep gradient using mobile phase A: 15 mM ammonium formate with 0.1% formic acid in water, and mobile phase B: acetonitrile. First, from 0 min to 4 min, the mobile phase consisted of 80% B isocratic flow; next, from 4 min to 10 min, the mobile phase consisted of a linear gradient changing from 80% to 99% B; from 10 min to 30 min, the mobile phase consisted of 99% B isocratic
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Fig. 3. LCMS chromatogram of an extract of human liver microsomes incubated with d6-αTQ-BSA and NADPH, showing only peaks with m/z 171 fragment ions. The process by which the peaks were identified is presented below.
XCalibur software (Thermo Scientific). Full scan of ions m/z 436, 445, and 451 were used to quantify d6-αTQ, d0-αHTQ, and d6-αHTQ, respectively. GC-MS analysis was performed either in Selected Ion mode (SIM) or scan mode using a Hewlett Packard 6890 gas chromatograph coupled to a Hewlett Packard 5872 mass selective detector. The gas chromatograph was fitted with a Hewlett Packard HP-1 methylsiloxane capillary column (30 m × 0.25 mm) and was operated in split injection mode isothermally at 285 °C using helium as the carrier gas. Post-reaction microsomal extracts were made as described above, the dried residue silylated under N2 with 40 μL each of pyridine and N,O-bis(trimethylsilyl)trifluoroacetamide/1% trimethylchlorosilane for 20 min at 65 °C. The silylated samples were then analyzed directly using a Hewlett Packard 6890 gas chromatograph coupled to a Hewlett Packard 5872 mass selective detector. The chromatogram was fitted with a Hewlett Packard HP-1 methylsiloxane capillary column (30 m × 0.25 mm) and was operated in split injection mode for 16 min in a 285° C oven using helium as the carrier gas.
We decided that the most rigorous way to test the stated hypothesis was to chemically synthesize the predicted CYP4F2 product, ω-OHαTQ, for use as an external standard. A chromatogram and mass spectrum of the synthesized standard, d0-OH-αTQ are shown in Fig. 4 A–B below. The mass spectrum of the 8.80 min peak in Fig. 3 is presented in Fig. 5. Both retention time and mass spectrum of the synthesized standard d0-OH-αTQ correspond with those of the 8.8 min peak of Fig. 3. As expected, the deuterium labeled OH-αTQ elutes slightly earlier than the unlabeled standard. In order to demonstrate NADPH-dependence of the synthesis of d6OH-αTQ from d6-αTQ, d6-αTQ-BSA was incubated in the microsomal system in the absence of NADPH; no d6-OH-αTQ was observed under these conditions, as shown in Fig. 6. Also missing from the negative control chromatogram, in comparison with the chromatogram of extracts of the full reaction system (Fig. 3, peaks with m/z 171 fragments) are the 7.1 min and 11.87 min peaks. The full mass spectrum of the 7.1 min peak, showing the m/z 171 fragment ion, along with the structure resulting from the mass spectrum interpretation, are shown in Fig. 7. To determine if the observed and identified ω-oxidation products of d6-αTQ were produced by the action of CYP4F2, microsomes from insect cells transfected with recombinant human CYP4F2 plus recombinant human CYP reductase and cytochrome b5, were incubated with d6-αTQ-BSA and NADPH as described above. These microsomes are devoid of all other P-450 enzymes. The LCMS chromatogram of an extract of that system is shown in Fig. 8. The 8.8 min peak and the 7.4 min peak were identified as d6-OHαTQ and d6-COOH-αTQ, respectively, by virtue of matching retention times and mass spectra to the d0-OH-αTQ standard (accounting for the six deuterium atoms), and the mass spectrum of the putative d6-carboxy-αTQ metabolite as described above for human liver microsomes. We consistently noticed a third peak in LCMS chromatograms of extracts of human liver microsomes incubated with d6-αTQ-BSA and NADPH that eluted at 11.9 min. (Fig. 3). The mass spectrum of that
3. Results We developed a method to measure d6-α-tocopheryl quinone (d6αTQ) and more polar reaction products by LCMS. The mass spectrum of the substrate d6-αTQ, and the fragmentation scheme is presented in Fig. 2A–B. Peaks at m/z 169 and 171 appear to be a fragment of the quinone and the hydroquinone, respectively. This suggests that there is significant reduction of the standard quinone sample after introduction to the MS source. This has been noted previously in the analysis of the naphthoquinone, vitamin K1, under electron impact ionization conditions (10). Note that the m/z 171 fragment includes the quinone ring portion of the molecule and its six deuterium atoms. A typical LCMS positive ESI full MS scan chromatogram of an extract of human liver microsomes incubated with d6-αTQ-BSA and NADPH, showed only peaks with m/z fragment 171 ions, is shown in Fig. 3. The large peak at 17 min corresponds to the retention time and mass spectrum of the substrate, d6-αTQ.
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A
B
Fig. 4. A: LCMS chromatogram of d0-OH-αTQ (std). B: LC mass spectrum of d0-OH-αTQ (std).
peak in shown in Fig. 9 and exhibited a strong m/z 171 fragment ion, suggesting it contained the deuterium-containing ring of the d6-αTQ substrate. The 11.9 min peak was first identified as d6-αT-hydroquinone on the basis of the mass spectrum. To confirm this identification, a solution of d6-αTQ in acetonitrile was treated with sodium borohydride crystals under N2 gas until the solution turned colorless. The solution was then immediately analyzed by LCMS under the same conditions as that of Fig. 3. The chromatogram showing extracted m/z 171 ions, and the complete mass spectrum of the colorless solution are shown below (Fig. 10). All of the findings reported here using LCMS were replicated using GCMS with the approach described in METHODS (data not shown).
4. Discussion The hypothesis under investigation was that α-tocopheryl quinone, the product of a 2-electron antioxidant function of α-tocopherol, undergoes an oxidative degradation pathway similar to that of α-tocopherol, and furthermore that this pathway is also initiated by the enzyme CYP4F2. The vitamin E ω-hydroxylase pathway begins with the CYP4F2catalyzed hydroxylation of one of the two terminal methyl groups in the hydrophobic side chain. This is followed by an oxidation to the terminal carboxyl; these two steps occur in the endoplasmic reticulum. Subsequent side chain shortening events, which probably occur in mitochondria7, terminate with the formation of 3′-carboxyethyl-hydroxychroman, or CEHC, which is glucuronidated and excreted in the
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Fig. 5. Mass spectrum of the 8.8 min peak in Fig. 3.
Fig. 6. LCMS chromatogram of extract of human liver microsomal system in the absence of NADPH (negative control), showing only peaks with m/z 171 ions.
urine. In mice this pathway occurs predominantly in the liver8. The presumed function of the tocopherol-ω-oxidation pathway is to eliminate excess or unwanted tocopherols and tocotrienols. Since CYP4F2 prefers forms of vitamin E other than α-tocopherol, it serves to minimize such forms in favor of α-tocopherol, resulting in the “alpha-tocopherol phenotype”. Until this report the metabolic fate of α-tocopheryl quinone (αTQ) was largely unknown. Herein we present convincing evidence that αTQ undergoes a similar ω-oxidation pathway as that of α-TOH, catalyzed by the same CYP enzyme, and as such can be included in the overall metabolic scheme as shown in Fig. 11. The presumed function of the tocopherol-ω-oxidation pathway is two-fold. Since CYP4F2 prefers forms of vitamin E other than α-
tocopherol, it serves to minimize such forms in favor of α-tocopherol, resulting in the “alpha-tocopherol phenotype”. Second, it serves to minimize the concentration of α-tocopherol in the endoplasmic reticulum of the liver, for reasons that are not understood. What is the physiological significance of the αTQ-ω-oxidation pathway? How important is this means of disposal of “spent” α-TOH? Measurement of αTQ content in rat heart9–11 and liver12,13 mitochondria show that there is about 20–100 fold less αTQ than αTOH in these membranes. αTQ has also been reported in human plasma at concentrations varying between 1000 and 5000-fold less than αTOH14–16. In atherosclerotic plaques the amount of αTQ is much higher, only ∼10 fold less than αTOH17,18. The increased amount of αTQ in plaques is not surprising since such lesions are known to be
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B
Fig. 7. A. Mass spectrum of the 7.1 min peak in Fig. 3. B: Proposed structure of the 7.1 min peak (d6-13′-CO2H-αTQ).
Fig. 8. LCMS chromatogram of extract of insect cell microsomes expressing only CYP4F2 and incubated with 10 μM d6-αTQ-BSA and NADPH.
the terminal product of the αTQ-ω-oxidation pathway. Pope et al.19 and Sharma et al.20 reported the definitive finding of α-tocopheronolactone glucuronide in human urine, using an authentic standard chemically synthesized for that purpose. However, the metabolic relationship between that excretory product and its putative precursor, αTQ, has been conjecture until now. The quantitative importance of the αTQ-ω-oxidation pathway in humans can be imputed from the data of Sharma et al.21, who reported
formed in part from oxidized LDL particles. αTQ formed in these sites would be expected to accumulate since endothelial cells are devoid of CYP4F2, thus the clearance of αTQ would be slow. The turnover of αTQ in plasma has not been reported. Its low concentration may reflect a low rate of formation from αTOH or a rapid rate of hepatic clearance via the CYP4F2-mediated pathway reported here. One indirect means of estimating whole-body catabolism of αTQ is through measuring urinary excretion of conjugated α-tocopheronolactone,
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Fig. 9. LC mass spectrum of the 11.9 min peak in Fig. 3.
mean urinary concentrations of conjugated α-tocopheronolactone in children with Type 1 diabetes, a disease considered one of elevated oxidative stress, was 15-fold higher than in non-diseased children. We used their metabolite concentration data, along with published values for daily creatinine excretion22 and daily αTOH intake in children23 to estimate that on average an astounding 85 percent of typical daily αtocopherol intake was excreted as conjugated metabolites of α-TQ (“Muller’s metabolites”) in the diabetic children as result of the irreversible oxidation of αTOH and the α-TQ ω-oxidation pathway described here. This calculation involved published estimates of daily αTOH intake and daily creatinine excretion in children of 6 mg αTOH/ day and 6.7 mmoles creatinine/day, respectively, since Sharma et al. measured neither value.
It must be recognized that the rate of excretion of conjugated αtocopheronolactone at any one time can potentially be influenced not only by the total body activity of CYP4F2 and the rate of lipid radical quenching by αTOH, but by the efficiency of recycling of αTO•. To date, none of these rates are directly measurable in humans. Regardless, identification of the 13-OH- and 13-COOH-metabolites of α-TQ and demonstration that they can be directly produced from α-TQ by CYP4F2 brings an important new dimension to the understanding of vitamin E turnover via oxidative metabolism and antioxidant function. Further, we demonstrate the quantitative significance of the αTQ-ωoxidation pathway in humans with conditions of elevated oxidative stress.
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Fig. 10. LCMS chromatogram (A) and mass spectrum (B) of the NaBH4 reduction product of d6-αTQ.
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Fig. 11. Overall scheme of vitamin E metabolism, showing the dual function of CYP4F2.
Acknowledgments
3. Sontag TJ, Parker RS. Cytochrome P450 omega-hydroxylase pathway of tocopherol catabolism. Novel mechanism of regulation of vitamin E status. J Biol Chem. 2002;277:25290–25296. https://doi.org/10.1074/jbc.M201466200. 4. Mazzini F, Betti M, Netscher T, Galli F, Salvadori P. Configuration of the vitamin E analogue garcinoic acid extracted from Garcinia Kola seeds. Chirality. 2009;21:519–524. https://doi.org/10.1002/chir.20630. 5. Weichet J, Blaha L. Studien in Der Vitamin-K- Und Vitamin-E-Reihe. 15. Uber Die Synthese Der 2,6,10,14-Tetramethyl-14-Hydroxy-15-Hexadecensaure Und Verwandter Verbindungen. Collect Czech Chem Commun. 1966;31:2424–2433. https://doi.org/10.1135/cccc19662424. 6. Sontag TJ, Parker RS. Influence of major structural features of tocopherols and tocotrienols on their omega-oxidation by tocopherol-omega-hydroxylase. J Lipid Res. 2007;48:1090–1098. https://doi.org/10.1194/jlr.M600514-JLR200. 7. Mustacich DJ, Leonard SW, Patel NK, Traber MG. α-Tocopherol β-oxidation localized to rat liver mitochondria. Free Radic Biol Med. 2010;48:73–81. https://doi.org/10. 1016/j.freeradbiomed.2009.10.024. 8. Bardowell SA, Ding X, Parker RS. Disruption of P450-mediated vitamin E hydroxylase activities alters vitamin E status in tocopherol supplemented mice and reveals extrahepatic vitamin E metabolism. J Lipid Res. 2012. https://doi.org/10.1194/jlr. M030734. 9. Leray C, Andriamampandry MD, Freund M, Gachet C, Cazenave JP. Simultaneous determination of homologues of vitamin E and coenzyme Q and products of alphatocopherol oxidation. J Lipid Res. 1998;39:2099–2105.
This work was supported by the Cornell University Department of Nutritional Sciences to RSP and by NSERC (RGPIN-155187 and RGPIN2017-06149) to JA. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmc.2018.10.002. References 1. Niki E. Role of vitamin E as a lipid-soluble peroxyl radical scavenger: in vitro and in vivo evidence. Free Radical Biol Med. 2014;66:3–12. https://doi.org/10.1016/j. freeradbiomed.2013.03.022. 2. Liebler DC, Burr JA. Oxidation of vitamin E during iron-catalyzed lipid peroxidation: evidence for electron-transfer reactions of the tocopheroxyl radical. Biochemistry. 1992;31:8278–8284https://DOI.org/10.1021/bi00150a022.
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L. Taylor et al. 10. Gregor W, Staniek K, Nohl H, Gille L. Distribution of tocopheryl quinone in mitochondrial membranes and interference with ubiquinone-mediated electron transfer. Biochem Pharmacol. 2006;71:1589–1601https://DOI.org/10.1016/j.bcp. 2006.02.012. 11. Staniek K, Rosenau T, Gregor W, Nohl H, Gille L. The protection of bioenergetic functions in mitochondria by new synthetic chromanols. Biochem Pharmacol. 2005;70:1361–1370. https://doi.org/10.1016/j.bcp.2005.07.030. 12. Gille L, Gregor W, Staniek K, Nohl H. Redox-interaction of alpha-tocopheryl quinone with isolated mitochondrial cytochrome bc1 complex. Biochem Pharmacol. 2004;68:373–381. https://doi.org/10.1016/j.bcp.2004.03.031. 13. Gille L, Staniek K, Rosenau T, Duvigneau JC, Kozlov AV. Tocopheryl quinones and mitochondria. Mol Nutr Food Res. 2010;54:601–615. https://doi.org/10.1002/mnfr. 200900386. 14. Mottier P, Gremaud E, Guy PA, Turesky RJ. Comparison of gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry methods to quantify alpha-tocopherol and alpha-tocopherolquinone levels in human plasma. Anal Biochem. 2002;301:128–135. https://doi.org/10.1006/abio.2001.5486. 15. Vatassery GT, Smith WE. Determination of alpha-tocopherolquinone (vitamin E quinone) in human serum, platelets, and red cell membrane samples. Anal Biochem. 1987;167:411–417. https://doi.org/10.1016/0003-2697(87)90185-0. 16. Jain SK, Wise R, Bocchini JJ. Vitamin E and vitamin E-quinone levels in red blood cells and plasma of newborn infants and their mothers. J Am Coll Nutr. 1996;15:44–48. https://doi.org/10.1080/07315724.1996.10718563.
17. Niu XW, Zammit V, Upston JM, Dean RT, Stocker R. Coexistence of oxidized lipids and alpha-tocopherol in all lipoprotein density fractions isolated from advanced human atherosclerotic plaques. Arteriosclerosis Thrombosis Vasc Biol. 1999;19:1708–1718. https://doi.org/10.1161/atvb.19.7.1708. 18. Stocker R, Keaney Jr JF. Role of oxidative modifications in atherosclerosis. Physiol Rev. 2004;84:1381–1478. https://doi.org/10.1152/physrev.00047.2003. 19. Pope SAS. The analysis and identification of urinary metabolites of vitamin E in man using mass spectrometry and chemical synthesis,. London, UK: University College London (University of London); 2001. 20. Sharma G, Muller D, O'Riordan S, et al. A novel method for the direct measurement of urinary conjugated metabolites of alpha-tocopherol and its use in diabetes. Mol Nutr Food Res. 2010;54:599–600. https://doi.org/10.1002/mnfr.200900378. 21. Sharma G, Muller DP, O'Riordan SM, et al. Urinary conjugated alpha-tocopheronolactone-a biomarker of oxidative stress in children with type 1 diabetes. Free Radic Biol Med. 2013;55:54–62. https://doi.org/10.1016/j.freeradbiomed.2012.09. 012. 22. Remer T, Neubert A, Maser-Gluth C. Anthropometry-based reference values for 24-h urinary creatinine excretion during growth and their use in endocrine and nutritional research. Am J Clin Nutr. 2002;75:561–569. https://doi.org/10.1093/ajcn/75.3.561. 23. Moshfegh A, Goldman J, Cleveland L. What We Eat in America, NHANES 2001–2002: Usual Nutrient Intakes from Food Compared to Dietary Reference Intakes., U.S. Department of Agriculture. Agric Res Serv. 2005.
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ω-Hydroxylation of α-tocopheryl quinone reveals a dual function for cytochrome P450-4F2 in vitamin E metabolism
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Luke Taylorb, Nick Kruegerb, Olga Malyshevaa, Jeffrey Atkinsonb, , Robert S. Parkera, ⁎
a b
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Division of Nutritional Sciences, Savage Hall, Cornell University, Ithaca, NY 14853, USA Department of Chemistry and Centre for Biotechnology, Brock University, 1812 Sir Isaac Brock Way, St. Catharines, Ontario L2S 3A1, Canada
ARTICLE INFO
ABSTRACT
This paper is dedicated to David Muller and Graham Burton, two giants of vitamin E biology.
α-Tocopherol (α-TOH) is the primary lipophilic radical trapping antioxidant in human tissues. Oxidative catabolism of α-tocopherol (αTOH) is initiated by ω-hydroxylation of the terminal carbon (C-13) of the isoprenoid sidechain followed by oxidative transformations that sequentially truncate the chain to yield the 2,5,7,8-tetramethyl(3′carboxyethyl)-6-hydroxychroman (α-CEHC). After conjugation to glucuronic acid, 3′-carboxyethyl-6hydroxychroman glucuronide is excreted in urine. We report here that the same enzyme that accomplishes this task, the cytochrome P450 monooxygenase CYP-4F2, can also ω-hydroxylate the terminal carbon of α-tocopheryl quinone. A standard sample of ω-OH-α-tocopheryl quinone (ω-OH-α-TQ) was synthesized as a mixture of stereoisomers by allylic oxidation of α-tocotrienol using SeO2 followed by double-bond reduction and oxidation to the quinone. After incubating human liver microsomes or insect cell microsomes expressing only recombinant human CYP-4F2, cytochrome b5, and NADPH P450 reductase with d6-α-tocopheryl quinone (d6-αTQ), we showed that the ω-hydroxylated (13-OH) d6-α-TQ was produced. We further identified the production of the terminal carboxylic acid d6-13-COOH-αTQ. The ramifications of this discovery to the understanding of tocopherol utilization and metabolism, including the quantitative importance of the αTQ-ω-hydroxylase pathway in humans, are discussed.
Keywords: Vitamin E α-Tocopherol α-Tocopheryl quinone Cytochrome P450-4F2 ω-Hydroxylation Oxidative metabolism
1. Introduction α-Tocopherol (αTOH) is the primary lipophilic antioxidant in cell membranes, lipoproteins, and lipid droplets. It functions by donating electrons to highly reactive lipid peroxides, thereby reducing them to less reactive lipid hydroperoxides. One mole of αTOH is capable of reducing two moles of lipid peroxide, first by using the phenolic hydrogen on the chromanol ring, forming the chromanoxyl radical of αTOH (αTO•). αTO• is a relatively stable radical, with a half-life of approximately 5 min in hexadecyltrimethylammonium chloride and milliseconds in DPPC bilayers (1, 2), and has two fates: (a) reduction to αTOH by small reducing substances such as reduced ubiquinone, ascorbic acid, and glutathione (see1 and references therein), or (b) reaction with a second lipid peroxyl radical (although see2 for alternative chemistries). This second reaction ultimately results in the irreversible oxidation of αTO• a to α-tocopheryl quinone (αTQ), as illustrated in Fig. 1 below. The metabolic fate of αTQ is at present unknown and is the subject of this investigation. Cytochrome P450-4F2 (CYP4F2) is an NADPH-dependent endoplasmic reticulum enzyme whose primary function appears to be the
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initiation of the oxidative degradation and elimination of tocopherols and tocotrienols3, thereby preventing their over-accumulation in cell membranes. CYP4F2 catalyzes the hydroxylation of one of the two terminal methyl groups on the tocochromanols, yielding (in the case of αTOH) ω-OH-αTOH. ω-OH-αTOH is then oxidized by a microsomal dehydrogenase to ω-COOH-αTOH. This intermediate then undergoes a series of oxidative chain-shortening reactions analogous to that of branched-chain fatty acids, ultimately yielding 2,5,7,8-tetramethyl (3′carboxyethyl)-6-hydroxychroman, or α-CEHC, which is glucuronidated and excreted in the urine, as illustrated in Fig. 11 in the Discussion section below. To date, CYP4F2 is the only enzyme proven to exhibit vitamin E-ω-hydroxylase activity. Sontag and Parker3 reported on the results of testing all available CYP enzymes (commercially available or gifts), including CYP3A4, for this activity. Only CYP4F2 tested positive. We hypothesized that αTQ undergoes a pathway of oxidative catabolism similar to that of αTOH, initiated by microsomal CYP4F2. In order to demonstrate this in the most rigorous fashion, we used deuterium-labeled αTQ as substrate and chemically synthesized the putative CYP4F2 reaction product, ω-OH-αTQ for use as a standard to probe
Corresponding authors. E-mail addresses: [email protected] (J. Atkinson), [email protected] (R.S. Parker).
https://doi.org/10.1016/j.bmc.2018.10.002 Received 24 August 2018; Received in revised form 28 September 2018; Accepted 4 October 2018 Available online 05 October 2018 0968-0896/ © 2018 Elsevier Ltd. All rights reserved.
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Fig. 1. Conversion of αTOH to αTQ by two-electron oxidation. After primary oxidation, αTO• may combine with a second peroxyl radical which is then hydrolyzed to yield αTQ. Alternatively, metal assisted single electron oxidation proceeds through the tocopherone cation2.
Scheme 1. Terminal allylic hydroxylation of α-tocotrienol acetate. (See Table 2 in Supplementary Information for variations in conditions and the corresponding isolated yields.).
formation of the deuterated metabolite by human liver microsomes and insect cell microsomes containing recombinant CYP4F2 and its reaction partners NADPH-CYP-reductase and cytochrome-b5.
microsomes (HLM) were purchased from Corning Incorporated (Corning NY). NADPH and NAD were purchased from Sigma-Aldrich Co. (St. Louis, MO). The microsomal reaction substrate d6-α-tocopherylquinone (d6αTQ) was synthesized in our lab via the FeCl3 oxidation of d6-α-TOH (a gift from Dr. Graham Burton) and used the in microsomal system as a bovine serum albumin (BSA) complex as described previously6.
2. Methods 2.1. Synthesis of d0-ω-hydroxy-α-tocopheryl quinone Having access to neither a convenient supply of garcinoic acid (from Garcinia kola)4 nor the desire to complete a multistep total synthesis5, we decided that it might be convenient to attempt a synthesis of a terminally hydroxylated α-tocopherol and its quinone by a selective terminal allylic oxidation of α-tocotrienol of which we had a large supply in hand. Catalytic reduction of the double bonds, reductive removal of the phenol protecting group and oxidation to the quinone would provide the desired ω-OH-α-tocopherylquinone (Scheme 1) Full synthetic and analytical data are provided in the Supplementary Material.
2.3. Microsomal reaction system Appropriate volumes of 4.4 mM d6-αTQ BSA complex were added to microsome suspensions consisting of either HLM (20 mg/ml protein) or CYP4F2 insect microsomes (100 pmol CYP/mL) and a sufficient volume of 100 mM KH2PO4 buffer pH 7.2, total volume 0.475 ml. This system was preincubated for 10 min in open glass culture tubes in a 37 °C water bath. NADPH and NAD (if used), final reaction concentrations of 1 mM, were added for a final reaction volume of 0.5 ml. Samples were incubated at 37 °C for an additional 20 min then immediately chilled on ice. d0-ω-Hydroxy-αTQ (the internal standard, in 5 μL ethanol) was then added and the microsomal system extracted first with 2 ml 95% ethyl acetate and then with 2 ml 1:1 hexanes:MTBE. The extracts were combined, evaporated to dryness under a stream of N2 and the residue dissolved in 100 μL acetonitrile for LCMS analysis, or in silylation reagents for GC-MS analysis.
2.2. Microsomes and deuterated αTQ substrate Recombinant insect cell microsomes expressing recombinant human CYP4F2, cytochrome b5, and NADPH P450 reductase, control insect cell microsomes devoid of all CYP enzymes, and pooled human liver
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A
B
Fig. 2. A: LC mass spectrum of d6-αTQ. B: Fragmentation of d6-αTQ, yielding the m/z 171 fragment ion.
2.4. LC-MS and GC-MS analysis of microsomal reaction products
flow; from 30 min to 35 min, the mobile phase consisted of a linear gradient changing from 99% to 80% B; finally, the column was equilibrated to initial conditions for another 5 min with 80%B. Flow rate was 0.3 ml/min. The Q Exactive mass spectrometer was equipped with a HESI probe and operated in positive ionization mode with parameters of: heater temperature, 30 °C; sheath gas, 40 (arbitrary units), auxiliary gas, 10 ml/min; sweep gas, 3 ml/min; spray voltage, 5 kV. Capillary temperature was set at 350 °C, and S-lens was 50. A full scan range was set at 50–670 (m/z). The resolution was set at 35,000. The maximum injection time (max IT) was 200 ms. Automated gain control (AGC) was targeted at 3 × 106 ions. For All Ion Fragmentation (AIF) analysis normalized energy collision energy (NCE) was 25%. Quantification of metabolites was performed using
UPLC/MS system Ultimate 3000 UHPLC (Dionex) coupled to Q Exactive-Mass spectrometer (QE-MS, Thermo Scientific, San Jose, CA) was used for chromatographic separation and peak analysis. Separation of d6 -a-tocopherol and metabolites was achieved by reversed phase liquid chromatography using an Acclaim RSLC 120 C18 column (2.1 × 150mm, 2.2 μm, Thermo Scientific) and multistep gradient using mobile phase A: 15 mM ammonium formate with 0.1% formic acid in water, and mobile phase B: acetonitrile. First, from 0 min to 4 min, the mobile phase consisted of 80% B isocratic flow; next, from 4 min to 10 min, the mobile phase consisted of a linear gradient changing from 80% to 99% B; from 10 min to 30 min, the mobile phase consisted of 99% B isocratic
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Fig. 3. LCMS chromatogram of an extract of human liver microsomes incubated with d6-αTQ-BSA and NADPH, showing only peaks with m/z 171 fragment ions. The process by which the peaks were identified is presented below.
XCalibur software (Thermo Scientific). Full scan of ions m/z 436, 445, and 451 were used to quantify d6-αTQ, d0-αHTQ, and d6-αHTQ, respectively. GC-MS analysis was performed either in Selected Ion mode (SIM) or scan mode using a Hewlett Packard 6890 gas chromatograph coupled to a Hewlett Packard 5872 mass selective detector. The gas chromatograph was fitted with a Hewlett Packard HP-1 methylsiloxane capillary column (30 m × 0.25 mm) and was operated in split injection mode isothermally at 285 °C using helium as the carrier gas. Post-reaction microsomal extracts were made as described above, the dried residue silylated under N2 with 40 μL each of pyridine and N,O-bis(trimethylsilyl)trifluoroacetamide/1% trimethylchlorosilane for 20 min at 65 °C. The silylated samples were then analyzed directly using a Hewlett Packard 6890 gas chromatograph coupled to a Hewlett Packard 5872 mass selective detector. The chromatogram was fitted with a Hewlett Packard HP-1 methylsiloxane capillary column (30 m × 0.25 mm) and was operated in split injection mode for 16 min in a 285° C oven using helium as the carrier gas.
We decided that the most rigorous way to test the stated hypothesis was to chemically synthesize the predicted CYP4F2 product, ω-OHαTQ, for use as an external standard. A chromatogram and mass spectrum of the synthesized standard, d0-OH-αTQ are shown in Fig. 4 A–B below. The mass spectrum of the 8.80 min peak in Fig. 3 is presented in Fig. 5. Both retention time and mass spectrum of the synthesized standard d0-OH-αTQ correspond with those of the 8.8 min peak of Fig. 3. As expected, the deuterium labeled OH-αTQ elutes slightly earlier than the unlabeled standard. In order to demonstrate NADPH-dependence of the synthesis of d6OH-αTQ from d6-αTQ, d6-αTQ-BSA was incubated in the microsomal system in the absence of NADPH; no d6-OH-αTQ was observed under these conditions, as shown in Fig. 6. Also missing from the negative control chromatogram, in comparison with the chromatogram of extracts of the full reaction system (Fig. 3, peaks with m/z 171 fragments) are the 7.1 min and 11.87 min peaks. The full mass spectrum of the 7.1 min peak, showing the m/z 171 fragment ion, along with the structure resulting from the mass spectrum interpretation, are shown in Fig. 7. To determine if the observed and identified ω-oxidation products of d6-αTQ were produced by the action of CYP4F2, microsomes from insect cells transfected with recombinant human CYP4F2 plus recombinant human CYP reductase and cytochrome b5, were incubated with d6-αTQ-BSA and NADPH as described above. These microsomes are devoid of all other P-450 enzymes. The LCMS chromatogram of an extract of that system is shown in Fig. 8. The 8.8 min peak and the 7.4 min peak were identified as d6-OHαTQ and d6-COOH-αTQ, respectively, by virtue of matching retention times and mass spectra to the d0-OH-αTQ standard (accounting for the six deuterium atoms), and the mass spectrum of the putative d6-carboxy-αTQ metabolite as described above for human liver microsomes. We consistently noticed a third peak in LCMS chromatograms of extracts of human liver microsomes incubated with d6-αTQ-BSA and NADPH that eluted at 11.9 min. (Fig. 3). The mass spectrum of that
3. Results We developed a method to measure d6-α-tocopheryl quinone (d6αTQ) and more polar reaction products by LCMS. The mass spectrum of the substrate d6-αTQ, and the fragmentation scheme is presented in Fig. 2A–B. Peaks at m/z 169 and 171 appear to be a fragment of the quinone and the hydroquinone, respectively. This suggests that there is significant reduction of the standard quinone sample after introduction to the MS source. This has been noted previously in the analysis of the naphthoquinone, vitamin K1, under electron impact ionization conditions (10). Note that the m/z 171 fragment includes the quinone ring portion of the molecule and its six deuterium atoms. A typical LCMS positive ESI full MS scan chromatogram of an extract of human liver microsomes incubated with d6-αTQ-BSA and NADPH, showed only peaks with m/z fragment 171 ions, is shown in Fig. 3. The large peak at 17 min corresponds to the retention time and mass spectrum of the substrate, d6-αTQ.
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A
B
Fig. 4. A: LCMS chromatogram of d0-OH-αTQ (std). B: LC mass spectrum of d0-OH-αTQ (std).
peak in shown in Fig. 9 and exhibited a strong m/z 171 fragment ion, suggesting it contained the deuterium-containing ring of the d6-αTQ substrate. The 11.9 min peak was first identified as d6-αT-hydroquinone on the basis of the mass spectrum. To confirm this identification, a solution of d6-αTQ in acetonitrile was treated with sodium borohydride crystals under N2 gas until the solution turned colorless. The solution was then immediately analyzed by LCMS under the same conditions as that of Fig. 3. The chromatogram showing extracted m/z 171 ions, and the complete mass spectrum of the colorless solution are shown below (Fig. 10). All of the findings reported here using LCMS were replicated using GCMS with the approach described in METHODS (data not shown).
4. Discussion The hypothesis under investigation was that α-tocopheryl quinone, the product of a 2-electron antioxidant function of α-tocopherol, undergoes an oxidative degradation pathway similar to that of α-tocopherol, and furthermore that this pathway is also initiated by the enzyme CYP4F2. The vitamin E ω-hydroxylase pathway begins with the CYP4F2catalyzed hydroxylation of one of the two terminal methyl groups in the hydrophobic side chain. This is followed by an oxidation to the terminal carboxyl; these two steps occur in the endoplasmic reticulum. Subsequent side chain shortening events, which probably occur in mitochondria7, terminate with the formation of 3′-carboxyethyl-hydroxychroman, or CEHC, which is glucuronidated and excreted in the
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Fig. 5. Mass spectrum of the 8.8 min peak in Fig. 3.
Fig. 6. LCMS chromatogram of extract of human liver microsomal system in the absence of NADPH (negative control), showing only peaks with m/z 171 ions.
urine. In mice this pathway occurs predominantly in the liver8. The presumed function of the tocopherol-ω-oxidation pathway is to eliminate excess or unwanted tocopherols and tocotrienols. Since CYP4F2 prefers forms of vitamin E other than α-tocopherol, it serves to minimize such forms in favor of α-tocopherol, resulting in the “alpha-tocopherol phenotype”. Until this report the metabolic fate of α-tocopheryl quinone (αTQ) was largely unknown. Herein we present convincing evidence that αTQ undergoes a similar ω-oxidation pathway as that of α-TOH, catalyzed by the same CYP enzyme, and as such can be included in the overall metabolic scheme as shown in Fig. 11. The presumed function of the tocopherol-ω-oxidation pathway is two-fold. Since CYP4F2 prefers forms of vitamin E other than α-
tocopherol, it serves to minimize such forms in favor of α-tocopherol, resulting in the “alpha-tocopherol phenotype”. Second, it serves to minimize the concentration of α-tocopherol in the endoplasmic reticulum of the liver, for reasons that are not understood. What is the physiological significance of the αTQ-ω-oxidation pathway? How important is this means of disposal of “spent” α-TOH? Measurement of αTQ content in rat heart9–11 and liver12,13 mitochondria show that there is about 20–100 fold less αTQ than αTOH in these membranes. αTQ has also been reported in human plasma at concentrations varying between 1000 and 5000-fold less than αTOH14–16. In atherosclerotic plaques the amount of αTQ is much higher, only ∼10 fold less than αTOH17,18. The increased amount of αTQ in plaques is not surprising since such lesions are known to be
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B
Fig. 7. A. Mass spectrum of the 7.1 min peak in Fig. 3. B: Proposed structure of the 7.1 min peak (d6-13′-CO2H-αTQ).
Fig. 8. LCMS chromatogram of extract of insect cell microsomes expressing only CYP4F2 and incubated with 10 μM d6-αTQ-BSA and NADPH.
the terminal product of the αTQ-ω-oxidation pathway. Pope et al.19 and Sharma et al.20 reported the definitive finding of α-tocopheronolactone glucuronide in human urine, using an authentic standard chemically synthesized for that purpose. However, the metabolic relationship between that excretory product and its putative precursor, αTQ, has been conjecture until now. The quantitative importance of the αTQ-ω-oxidation pathway in humans can be imputed from the data of Sharma et al.21, who reported
formed in part from oxidized LDL particles. αTQ formed in these sites would be expected to accumulate since endothelial cells are devoid of CYP4F2, thus the clearance of αTQ would be slow. The turnover of αTQ in plasma has not been reported. Its low concentration may reflect a low rate of formation from αTOH or a rapid rate of hepatic clearance via the CYP4F2-mediated pathway reported here. One indirect means of estimating whole-body catabolism of αTQ is through measuring urinary excretion of conjugated α-tocopheronolactone,
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Fig. 9. LC mass spectrum of the 11.9 min peak in Fig. 3.
mean urinary concentrations of conjugated α-tocopheronolactone in children with Type 1 diabetes, a disease considered one of elevated oxidative stress, was 15-fold higher than in non-diseased children. We used their metabolite concentration data, along with published values for daily creatinine excretion22 and daily αTOH intake in children23 to estimate that on average an astounding 85 percent of typical daily αtocopherol intake was excreted as conjugated metabolites of α-TQ (“Muller’s metabolites”) in the diabetic children as result of the irreversible oxidation of αTOH and the α-TQ ω-oxidation pathway described here. This calculation involved published estimates of daily αTOH intake and daily creatinine excretion in children of 6 mg αTOH/ day and 6.7 mmoles creatinine/day, respectively, since Sharma et al. measured neither value.
It must be recognized that the rate of excretion of conjugated αtocopheronolactone at any one time can potentially be influenced not only by the total body activity of CYP4F2 and the rate of lipid radical quenching by αTOH, but by the efficiency of recycling of αTO•. To date, none of these rates are directly measurable in humans. Regardless, identification of the 13-OH- and 13-COOH-metabolites of α-TQ and demonstration that they can be directly produced from α-TQ by CYP4F2 brings an important new dimension to the understanding of vitamin E turnover via oxidative metabolism and antioxidant function. Further, we demonstrate the quantitative significance of the αTQ-ωoxidation pathway in humans with conditions of elevated oxidative stress.
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Fig. 10. LCMS chromatogram (A) and mass spectrum (B) of the NaBH4 reduction product of d6-αTQ.
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Fig. 11. Overall scheme of vitamin E metabolism, showing the dual function of CYP4F2.
Acknowledgments
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This work was supported by the Cornell University Department of Nutritional Sciences to RSP and by NSERC (RGPIN-155187 and RGPIN2017-06149) to JA. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmc.2018.10.002. References 1. Niki E. Role of vitamin E as a lipid-soluble peroxyl radical scavenger: in vitro and in vivo evidence. Free Radical Biol Med. 2014;66:3–12. https://doi.org/10.1016/j. freeradbiomed.2013.03.022. 2. Liebler DC, Burr JA. Oxidation of vitamin E during iron-catalyzed lipid peroxidation: evidence for electron-transfer reactions of the tocopheroxyl radical. Biochemistry. 1992;31:8278–8284https://DOI.org/10.1021/bi00150a022.
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17. Niu XW, Zammit V, Upston JM, Dean RT, Stocker R. Coexistence of oxidized lipids and alpha-tocopherol in all lipoprotein density fractions isolated from advanced human atherosclerotic plaques. Arteriosclerosis Thrombosis Vasc Biol. 1999;19:1708–1718. https://doi.org/10.1161/atvb.19.7.1708. 18. Stocker R, Keaney Jr JF. Role of oxidative modifications in atherosclerosis. Physiol Rev. 2004;84:1381–1478. https://doi.org/10.1152/physrev.00047.2003. 19. Pope SAS. The analysis and identification of urinary metabolites of vitamin E in man using mass spectrometry and chemical synthesis,. London, UK: University College London (University of London); 2001. 20. Sharma G, Muller D, O'Riordan S, et al. A novel method for the direct measurement of urinary conjugated metabolites of alpha-tocopherol and its use in diabetes. Mol Nutr Food Res. 2010;54:599–600. https://doi.org/10.1002/mnfr.200900378. 21. Sharma G, Muller DP, O'Riordan SM, et al. Urinary conjugated alpha-tocopheronolactone-a biomarker of oxidative stress in children with type 1 diabetes. Free Radic Biol Med. 2013;55:54–62. https://doi.org/10.1016/j.freeradbiomed.2012.09. 012. 22. Remer T, Neubert A, Maser-Gluth C. Anthropometry-based reference values for 24-h urinary creatinine excretion during growth and their use in endocrine and nutritional research. Am J Clin Nutr. 2002;75:561–569. https://doi.org/10.1093/ajcn/75.3.561. 23. Moshfegh A, Goldman J, Cleveland L. What We Eat in America, NHANES 2001–2002: Usual Nutrient Intakes from Food Compared to Dietary Reference Intakes., U.S. Department of Agriculture. Agric Res Serv. 2005.
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