Analysis of vitamin E metabolites including carboxychromanols and sulfated derivatives using LC/MS/MS

Abstract

Tocopherols and tocotrienols are metabolized via hydroxylation and oxidation of their hydrophobic side chain to generate 13′-hydroxychromanols (13′-OHs) and various carboxychromanols, which can be further metabolized by conjugation including sulfation. Recent studies indicate that long-chain carboxychromanols, especially 13′-carboxychromanol (13′-COOH), appear to be more bioactive than tocopherols in anti-inflammatory and anticancer actions. To understand the potential contribution of metabolites to vitamin E-mediated effects, an accurate assay is needed to evaluate bioavailability of these metabolites. Here we describe an LC/MS/MS assay for quantifying vitamin E metabolites using negative polarity ESI. This assay includes a reliable sample extraction procedure with efficacy of ≥ 89% and interday/intraday variation of 3–11% for major metabolites. To ensure accurate quantification, short-chain, long-chain, and sulfated carboxychromanols are included as external/internal standards. Using this assay, we observed that sulfated carboxychromanols are the primary metabolites in the plasma of rodents fed with γ-tocopherol or δ-tocopherol. Although plasma levels of 13′-COOHs and 13′-OHs are low, high concentrations of these compounds are found in feces. Our study demonstrates an LC/MS/MS assay for quantitation of sulfated and unconjugated vitamin E metabolites, and this assay will be useful for evaluating the role of these metabolites in vivo. Tocopherols and tocotrienols are metabolized via hydroxylation and oxidation of their hydrophobic side chain to generate 13′-hydroxychromanols (13′-OHs) and various carboxychromanols, which can be further metabolized by conjugation including sulfation. Recent studies indicate that long-chain carboxychromanols, especially 13′-carboxychromanol (13′-COOH), appear to be more bioactive than tocopherols in anti-inflammatory and anticancer actions. To understand the potential contribution of metabolites to vitamin E-mediated effects, an accurate assay is needed to evaluate bioavailability of these metabolites. Here we describe an LC/MS/MS assay for quantifying vitamin E metabolites using negative polarity ESI. This assay includes a reliable sample extraction procedure with efficacy of ≥ 89% and interday/intraday variation of 3–11% for major metabolites. To ensure accurate quantification, short-chain, long-chain, and sulfated carboxychromanols are included as external/internal standards. Using this assay, we observed that sulfated carboxychromanols are the primary metabolites in the plasma of rodents fed with γ-tocopherol or δ-tocopherol. Although plasma levels of 13′-COOHs and 13′-OHs are low, high concentrations of these compounds are found in feces. Our study demonstrates an LC/MS/MS assay for quantitation of sulfated and unconjugated vitamin E metabolites, and this assay will be useful for evaluating the role of these metabolites in vivo. The vitamin E family comprises eight lipophilic antioxidants, α-, β-, γ- and δ-tocopherol (αT, βT, γT, and δT) and the corresponding tocotrienols (αTE, βTE, γTE, and δTE) (Fig. 1). Among the members of the vitamin E family, αT is the predominant form in tissues and is preferentially bound to a tocopherol transport protein that prevents αT from being extensively metabolized (1Jiang Q. Natural forms of vitamin E: metabolism, antioxidant, and anti-inflammatory activities and their role in disease prevention and therapy.Free Radic. Biol. Med. 2014; 72: 76-90Crossref PubMed Scopus (518) Google Scholar). In contrast, other vitamin E forms are largely metabolized in the liver via ω-hydr­oxylation and oxidation to generate 13′-hydroxychromanol (13′-OH) and 13′-carboxychromanol (13′-COOH), the latter of which is subsequently metabolized via β-oxidation to various shorter-chain carboxychromanols including the terminal metabolite observed in urine, 3′-COOH [or 2-(β-carboxyethyl)-6-hydroxychroman (CEHC)] (Fig. 2) (1Jiang Q. Natural forms of vitamin E: metabolism, antioxidant, and anti-inflammatory activities and their role in disease prevention and therapy.Free Radic. Biol. Med. 2014; 72: 76-90Crossref PubMed Scopus (518) Google Scholar, 2Birringer M. Pfluger P. Kluth D. Landes N. Brigelius-Flohe R. Identities and differences in the metabolism of tocotrienols and tocopherols in HepG2 cells.J. Nutr. 2002; 132: 3113-3118Crossref PubMed Scopus (183) Google Scholar, 3Sontag T.J. Parker R.S. Cytochrome P450 omega-hydroxylase pathway of tocopherol catabolism. Novel mechanism of regulation of vitamin E status.J. Biol. Chem. 2002; 277: 25290-25296Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). In parallel with β-oxidation, long-chain carboxychromanols and CEHCs appear to be conjugated via sulfation to form conjugated carboxychromanols (Fig. 2) (4Jiang Q. Freiser H. Wood K.V. Yin X. Identification and quantitation of novel vitamin E metabolites, sulfated long-chain carboxychromanols, in human A549 cells and in rats.J. Lipid Res. 2007; 48: 1221-1230Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 5Freiser H. Jiang Q. Gamma-tocotrienol and gamma-tocopherol are primarily metabolized to conjugated 2-(beta-carboxyethyl)-6-hydroxy-2,7,8-trimethylchroman and sulfated long-chain carboxychromanols in rats.J. Nutr. 2009; 139: 884-889Crossref PubMed Scopus (57) Google Scholar). The terminal metabolite CEHCs and their conjugated forms are found in the urine (6Chiku S. Hamamura K. Nakamura T. Novel urinary metabolite of d-delta-tocopherol in rats.J. Lipid Res. 1984; 25: 40-48Abstract Full Text PDF PubMed Google Scholar, 7Schultz M. Leist M. Petrzika M. Gassmann B. Brigelius-Flohe R. Novel urinary metabolite of alpha-tocopherol, 2,5,7,8-tetramethyl-2(2′-carboxyethyl)-6-hydroxychroman, as an indicator of an adequate vitamin E supply?.Am. J. Clin. Nutr. 1995; 62: 1527S-1534SCrossref PubMed Google Scholar, 8Traber M.G. Elsner A. Brigelius-Flohe R. Synthetic as compared with natural vitamin E is preferentially excreted as alpha-CEHC in human urine: studies using deuterated alpha-tocopheryl acetates.FEBS Lett. 1998; 437: 145-148Crossref PubMed Scopus (116) Google Scholar, 9Swanson J.E. Ben R.N. Burton G.W. Parker R.S. Urinary excretion of 2,7, 8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman is a major route of elimination of gamma-tocopherol in humans.J. Lipid Res. 1999; 40: 665-671Abstract Full Text Full Text PDF PubMed Google Scholar, 10Stahl W. Graf P. Brigelius-Flohe R. Wechter W. Sies H. Quantification of the alpha- and gamma-tocopherol metabolites 2,5,7, 8-tetramethyl-2-(2′-carboxyethyl)-6-hydroxychroman and 2,7, 8-trimethyl-2-(2′-carboxyethyl)-6-hydroxychroman in human serum.Anal. Biochem. 1999; 275: 254-259Crossref PubMed Scopus (86) Google Scholar, 11Pope S.A. Burtin G.E. Clayton P.T. Madge D.J. Muller D.P. Synthesis and analysis of conjugates of the major vitamin E metabolite, alpha-CEHC.Free Radic. Biol. Med. 2002; 33: 807-817Crossref PubMed Scopus (60) Google Scholar). On the other hand, unconjugated short-, mid-, and long-chain carboxychromanols have recently been detected in feces after supplementation of γT or δT (12Jiang Q. Jiang Z. Hall Y.J. Jang Y. Snyder P.W. Bain C. Huang J. Jannasch A. Cooper B. Wang Y. et al.Gamma-tocopherol attenuates moderate but not severe colitis and suppresses moderate colitis-promoted colon tumorigenesis in mice.Free Radic. Biol. Med. 2013; 65: 1069-1077Crossref PubMed Scopus (37) Google Scholar, 13Bardowell S.A. Ding X. Parker R.S. Disruption of P450-mediated vitamin E hydroxylase activities alters vitamin E status in tocopherol supplemented mice and reveals extra-hepatic vitamin E metabolism.J. Lipid Res. 2012; 53: 2667-2676Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 14Bardowell S.A. Duan F. Manor D. Swanson J.E. Parker R.S. Disruption of mouse cytochrome p450 4f14 (Cyp4f14 gene) causes severe perturbations in vitamin E metabolism.J. Biol. Chem. 2012; 287: 26077-26086Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar).Fig. 2Vitamin E (γT) metabolism and metabolites. Vitamin E forms are metabolized via ω-hydroxylation and oxidation to generate 13′-OH and 13′-COOH, which is subsequently metabolized via β-oxidation to form 11′-, 9′-, 7′-, 5′-, and 3′-COOH. Sulfation of intermediate carboxychromanols appears to take place in parallel with β-oxidation in response to supplementation of vitamin E forms.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although research on vitamin E has predominantly focused on tocopherols and tocotrienols, their metabolites have been shown to have biological activities that are stronger than or differ from the unmetabolized vitamins. For instance, γ-CEHC has been reported to have natriuratic activity in vitro and in animals (15Wechter W.J. Kantoci D. Murray Jr., E.D. D'Amico D.C. Jung M.E. Wang W.H. A new endogenous natriuretic factor: LLU-α.Proc. Natl. Acad. Sci. USA. 1996; 93: 6002-6007Crossref PubMed Scopus (170) Google Scholar). Recent studies including ours have demonstrated that long-chain carboxychromanols appear to have unique anti-inflammatory effects by inhibition of cyclooxygenases and 5-lipoxygenase and induced apoptosis in cancer cells (16Jiang Q. Yin X. Lill M.A. Danielson M.L. Freiser H. Huang J. Long-chain carboxychromanols, metabolites of vitamin E, are potent inhibitors of cyclooxygenases.Proc. Natl. Acad. Sci. USA. 2008; 105: 20464-20469Crossref PubMed Scopus (147) Google Scholar, 17Jiang Z. Yin X. Jiang Q. Natural forms of vitamin E and 13′-carboxychromanol, a long-chain vitamin E metabolite, inhibit leukotriene generation from stimulated neutrophils by blocking calcium influx and suppressing 5-lipoxygenase activity, respectively.J. Immunol. 2011; 186: 1173-1179Crossref PubMed Scopus Google Scholar, M. D. S. S. D. M. M. effects of long-chain vitamin E metabolites in HepG2 cells are by Radic. Biol. Med. PubMed Scopus Google Scholar). For these long-chain carboxychromanols are stronger than tocopherols or shorter-chain these of vitamin E metabolites, their role in vitamin E-mediated effects of the is that is the bioavailability of these metabolites in To this a reliable and accurate is needed to evaluate these metabolites can be in the Although we have an assay for various vitamin E metabolites (4Jiang Q. Freiser H. Wood K.V. Yin X. Identification and quantitation of novel vitamin E metabolites, sulfated long-chain carboxychromanols, in human A549 cells and in rats.J. Lipid Res. 2007; 48: 1221-1230Abstract Full Text Full Text PDF PubMed Scopus (57) Google this is not for quantifying metabolites as a of supplementation of a of vitamin E are not not sulfated carboxychromanols and not have for extraction and M.G. of liver vitamin E metabolites by vitamin E Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. C. B. of metabolites of tocopherols and tocotrienols in mice and humans.J. Chem. PubMed Scopus Google Scholar). an assay using being is not of conjugated metabolites J.E. Ben R.N. Burton G.W. Parker R.S. Urinary excretion of 2,7, 8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman is a major route of elimination of gamma-tocopherol in humans.J. Lipid Res. 1999; 40: 665-671Abstract Full Text Full Text PDF PubMed Google Scholar). In this we describe a LC/MS/MS assay that carboxychromanols, and sulfated metabolites. To ensure accurate assay includes an sample and various carboxychromanols and a sulfated as and standards. this assay to bioavailability of vitamin E metabolites in urine, and from rodents supplemented with γT or αT γT and δT from γ-CEHC and from and which are metabolites from δT and to a procedure D.J. A of Lett. PubMed Scopus (37) Google Scholar). from other from The human A549 cells from in with For cells cells in with and for or (4Jiang Q. Freiser H. Wood K.V. Yin X. Identification and quantitation of novel vitamin E metabolites, sulfated long-chain carboxychromanols, in human A549 cells and in rats.J. Lipid Res. 2007; 48: 1221-1230Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). and in long-chain carboxychromanols and sulfated metabolites (4Jiang Q. Freiser H. Wood K.V. Yin X. Identification and quantitation of novel vitamin E metabolites, sulfated long-chain carboxychromanols, in human A549 cells and in rats.J. Lipid Res. 2007; 48: 1221-1230Abstract Full Text Full Text PDF PubMed Scopus (57) Google for LC/MS/MS or by a of and of via for as an for the in a new with The a and the more with of and the and in being by or the extraction from LC/MS/MS or as an of in of with of and in which with of as being by of urine with and by of The extraction more with of The This procedure of (4Jiang Q. Freiser H. Wood K.V. Yin X. Identification and quantitation of novel vitamin E metabolites, sulfated long-chain carboxychromanols, in human A549 cells and in rats.J. Lipid Res. 2007; 48: 1221-1230Abstract Full Text Full Text PDF PubMed Scopus (57) Google vitamin E metabolites, and tocotrienols on a using and detected by a with the and and This in in the extraction efficacy and of metabolites and vitamin E The LC/MS/MS analysis with an to an with a The an from A of and of which with to to The as B. The with a of to polarity with the and an of with by from by of with A549 cells for as (4Jiang Q. Freiser H. Wood K.V. Yin X. Identification and quantitation of novel vitamin E metabolites, sulfated long-chain carboxychromanols, in human A549 cells and in rats.J. Lipid Res. 2007; 48: 1221-1230Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). To the of to to H. Jiang Q. of the and analysis of plasma conjugated and sulfated long-chain carboxychromanols, metabolites of vitamin Biochem. 2009; PubMed Scopus Google which using with is as an for quantifying sulfated long-chain metabolites. the studies by and In the study of metabolite in response to a of γT or from in for a for and by with γT or δT by using as the in animals of animals and and other tissues In γT by and urine for The urine and In the study of metabolite in response to or mice from and with to and a of mice and or mice to of colon tumorigenesis by as (12Jiang Q. Jiang Z. Hall Y.J. Jang Y. Snyder P.W. Bain C. Huang J. Jannasch A. Cooper B. Wang Y. et al.Gamma-tocopherol attenuates moderate but not severe colitis and suppresses moderate colitis-promoted colon tumorigenesis in mice.Free Radic. Biol. Med. 2013; 65: 1069-1077Crossref PubMed Scopus (37) Google Scholar). the study mice on supplemented for more than (12Jiang Q. Jiang Z. Hall Y.J. Jang Y. Snyder P.W. Bain C. Huang J. Jannasch A. Cooper B. Wang Y. et al.Gamma-tocopherol attenuates moderate but not severe colitis and suppresses moderate colitis-promoted colon tumorigenesis in mice.Free Radic. Biol. Med. 2013; 65: 1069-1077Crossref PubMed Scopus (37) Google Scholar). plasma and feces In the study of extraction to and in the for of with are as In vitamin E metabolites after sample to This has been shown to or carboxychromanols as and (4Jiang Q. Freiser H. Wood K.V. Yin X. Identification and quantitation of novel vitamin E metabolites, sulfated long-chain carboxychromanols, in human A549 cells and in rats.J. Lipid Res. 2007; 48: 1221-1230Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). extraction for 13′-COOHs by this procedure not especially for plasma or is that vitamin E forms and their metabolites to be by which For these we to and the extraction procedure to vitamin E forms and the metabolites from biological To and evaluate extraction we with vitamin E forms and metabolites including γT, and The extraction procedure included an extraction with a of and of we and an extraction of the with in and The tocopherols and tocotrienols, and the carboxychromanols and This procedure high extraction and as in on efficacy and sample of Tocopherols and metabolites to γ-CEHC γT and δT of by and via for for the a with The a and the with for the The and The and in being by with are as on in a new of Tocopherols and metabolites to γ-CEHC γT and δT of by and via for for the a with The a and the with for the The and The and in being by with are as on the of we compared efficacy of carboxychromanols including and and In these we and γ-CEHC as the compounds and found that in the negative stronger than other the by and for and To of other metabolites including sulfated we that sulfated carboxychromanols by of A549 cells with (4Jiang Q. Freiser H. Wood K.V. Yin X. Identification and quantitation of novel vitamin E metabolites, sulfated long-chain carboxychromanols, in human A549 cells and in rats.J. Lipid Res. 2007; 48: 1221-1230Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 5Freiser H. Jiang Q. Gamma-tocotrienol and gamma-tocopherol are primarily metabolized to conjugated 2-(beta-carboxyethyl)-6-hydroxy-2,7,8-trimethylchroman and sulfated long-chain carboxychromanols in rats.J. Nutr. 2009; 139: 884-889Crossref PubMed Scopus (57) Google Scholar). of carboxychromanols are shown in of various vitamin E metabolites by of the side chain with in the of the (Fig. of the observed for metabolites with a of the (Fig. To we the the for for quantifying The for carboxychromanols and vitamin E forms are in for vitamin E the are using negative The with have been by the in a new the are using negative The with have been by the be that sulfated metabolites, we to carboxychromanols using on a or and by and we not of metabolites in the plasma of with carboxychromanols have been after being to unconjugated by H. Jiang Q. of the and analysis of plasma conjugated and sulfated long-chain carboxychromanols, metabolites of vitamin Biochem. 2009; PubMed Scopus Google Scholar). this not the of conjugation and be sample is indicate that or not to long-chain carboxychromanols as In to sulfated metabolites, we from from cells with (16Jiang Q. Yin X. Lill M.A. Danielson M.L. Freiser H. Huang J. Long-chain carboxychromanols, metabolites of vitamin E, are potent inhibitors of cyclooxygenases.Proc. Natl. Acad. Sci. USA. 2008; 105: 20464-20469Crossref PubMed Scopus (147) Google and The of by with after being to with have subsequently as an to sulfated long-chain observed a of LC/MS/MS response and concentrations to for and long-chain metabolites including and the for carboxychromanols on with a of To efficacy in extraction of and to the of or plasma that by with as an the found that these compounds in the plasma or to in that the extraction not have on quantitation of metabolites using with efficacy carboxychromanols can from to is to these compounds by and that are with biological in the analysis of biological we and to 13′-OHs and long-chain carboxychromanols, for sulfated long-chain metabolites, and γ-CEHC or for In these and as and the as standards. that long-chain metabolites of the For instance, and have the these metabolites differ in will be for of these compounds K. X. of by a in 2011; PubMed Scopus Google Scholar). Using the LC/MS/MS assay, we found that mice fed a supplemented with γT or δT but of and sulfated long-chain carboxychromanols as as in the of these metabolites in mice fed the in to concentrations of metabolites in the we found high levels of unconjugated short-, mid-, and in feces. Among excretion metabolites, and 13′-OH the predominant vitamin E metabolites in the plasma of mice fed or mice fed or or for and these mice to and for and for LC/MS/MS or the are as and supplemented in a new detected in from mice fed supplemented with γT or δT from the study in or are as and supplemented in a new mice fed or or for and these mice to and for and for LC/MS/MS or the are as and supplemented from the study in or are as and supplemented To evaluate the bioavailability of metabolites as a of a high of we a of γT or δT and plasma found that sulfated CEHCs and the major metabolites in the plasma of by with (4Jiang Q. Freiser H. Wood K.V. Yin X. Identification and quantitation of novel vitamin E metabolites, sulfated long-chain carboxychromanols, in human A549 cells and in rats.J. Lipid Res. 2007; 48: 1221-1230Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 5Freiser H. Jiang Q. Gamma-tocotrienol and gamma-tocopherol are primarily metabolized to conjugated 2-(beta-carboxyethyl)-6-hydroxy-2,7,8-trimethylchroman and sulfated long-chain carboxychromanols in rats.J. Nutr. 2009; 139: 884-889Crossref PubMed Scopus (57) Google the LC/MS/MS of sulfated with on in conjugated carboxychromanols in the plasma the of sample detected in the plasma of after a of γT or δT a of γT or δT in or animals and plasma and on sample and the LC/MS/MS are in and or the are as and tocopherol in a new a of γT or δT in or animals and plasma and on sample and the LC/MS/MS are in and or the are as and tocopherol In a we urine after with γT or observed high excretion of sulfated γ-CEHC in but not of γT to of sulfated γ-CEHC and unconjugated form to and have a and LC/MS/MS assay for carboxychromanols, and sulfated vitamin E metabolites. Our is by of major metabolites and an extraction procedure that in high and have carboxychromanols as and to ensure accurate In has a as a which of sulfated metabolites the for a for the the of sulfated CEHCs in the The to conjugated metabolites including sulfated and more accurate quantitation of these compounds than with to the Our indicate that with not the from sulfated 13′-COOH, conjugated and can be to their unconjugated via H. Jiang Q. of the and analysis of plasma conjugated and sulfated long-chain carboxychromanols, metabolites of vitamin Biochem. 2009; PubMed Scopus Google Scholar). This is sulfated long-chain metabolites are for the a the with can conjugated carboxychromanols, but not the long-chain Our assay accurate and quantitation of and using as a sulfated and metabolites have this an to of conjugates and be useful for further of their in vivo. On the other hand, to carboxychromanols we not of these metabolites in the plasma of with to be carboxychromanols can be in other or human Using the LC/MS/MS assay, we found of vitamin E metabolite in response to supplementation of γT or of γT or δT via a to plasma concentrations of sulfated carboxychromanols than the concentrations via with This can be by the that animals a of γT or δT from supplemented than a by which tocopherols as a high plasma levels of sulfated metabolites detected after that sulfation of long-chain or intermediate carboxychromanols place in parallel with β-oxidation a of tocopherols is of unconjugated metabolites, especially and which are in the are detected in feces. This is with (12Jiang Q. Jiang Z. Hall Y.J. Jang Y. Snyder P.W. Bain C. Huang J. Jannasch A. Cooper B. Wang Y. et al.Gamma-tocopherol attenuates moderate but not severe colitis and suppresses moderate colitis-promoted colon tumorigenesis in mice.Free Radic. Biol. Med. 2013; 65: 1069-1077Crossref PubMed Scopus (37) Google Scholar, 13Bardowell S.A. Ding X. Parker R.S. Disruption of P450-mediated vitamin E hydroxylase activities alters vitamin E status in tocopherol supplemented mice and reveals extra-hepatic vitamin E metabolism.J. Lipid Res. 2012; 53: 2667-2676Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 14Bardowell S.A. Duan F. Manor D. Swanson J.E. Parker R.S. Disruption of mouse cytochrome p450 4f14 (Cyp4f14 gene) causes severe perturbations in vitamin E metabolism.J. Biol. Chem. 2012; 287: 26077-26086Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The high of carboxychromanols in feces that long-chain metabolites are not to the after being in the but are excreted via the has recently been that to of vitamin E metabolites be excreted to feces S.A. Ding X. Parker R.S. Disruption of P450-mediated vitamin E hydroxylase activities alters vitamin E status in tocopherol supplemented mice and reveals extra-hepatic vitamin E metabolism.J. Lipid Res. 2012; 53: 2667-2676Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 14Bardowell S.A. Duan F. Manor D. Swanson J.E. Parker R.S. Disruption of mouse cytochrome p450 4f14 (Cyp4f14 gene) causes severe perturbations in vitamin E metabolism.J. Biol. Chem. 2012; 287: 26077-26086Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). is that high levels of these metabolites in feces are by an further with that and 13′-OH are the major excretion metabolites of vitamin In conjugated CEHCs have been reported in the urine (6Chiku S. Hamamura K. Nakamura T. Novel urinary metabolite of d-delta-tocopherol in rats.J. Lipid Res. 1984; 25: 40-48Abstract Full Text PDF PubMed Google Scholar, 7Schultz M. Leist M. Petrzika M. Gassmann B. Brigelius-Flohe R. Novel urinary metabolite of alpha-tocopherol, 2,5,7,8-tetramethyl-2(2′-carboxyethyl)-6-hydroxychroman, as an indicator of an adequate vitamin E supply?.Am. J. Clin. Nutr. 1995; 62: 1527S-1534SCrossref PubMed Google Scholar, 8Traber M.G. Elsner A. Brigelius-Flohe R. Synthetic as compared with natural vitamin E is preferentially excreted as alpha-CEHC in human urine: studies using deuterated alpha-tocopheryl acetates.FEBS Lett. 1998; 437: 145-148Crossref PubMed Scopus (116) Google Scholar, 9Swanson J.E. Ben R.N. Burton G.W. Parker R.S. Urinary excretion of 2,7, 8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman is a major route of elimination of gamma-tocopherol in humans.J. Lipid Res. 1999; 40: 665-671Abstract Full Text Full Text PDF PubMed Google Scholar, 10Stahl W. Graf P. Brigelius-Flohe R. Wechter W. Sies H. Quantification of the alpha- and gamma-tocopherol metabolites 2,5,7, 8-tetramethyl-2-(2′-carboxyethyl)-6-hydroxychroman and 2,7, 8-trimethyl-2-(2′-carboxyethyl)-6-hydroxychroman in human serum.Anal. Biochem. 1999; 275: 254-259Crossref PubMed Scopus (86) Google Scholar, 11Pope S.A. Burtin G.E. Clayton P.T. Madge D.J. Muller D.P. Synthesis and analysis of conjugates of the major vitamin E metabolite, alpha-CEHC.Free Radic. Biol. Med. 2002; 33: 807-817Crossref PubMed Scopus (60) Google we sulfated are of in the urine of that fed of has been demonstrated that vitamin E metabolites are more bioactive or have activities compared with unmetabolized vitamins. but not has natriuretic activity (15Wechter W.J. Kantoci D. Murray Jr., E.D. D'Amico D.C. Jung M.E. Wang W.H. A new endogenous natriuretic factor: LLU-α.Proc. Natl. Acad. Sci. USA. 1996; 93: 6002-6007Crossref PubMed Scopus (170) Google and is to be for excretion in response to supplementation of in rodents H. C. H. T. K. a vitamin E is a natriuretic Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). have demonstrated that 13′-COOHs are inhibitors of of the tocopherols or sulfated long-chain metabolites inhibit the activity (16Jiang Q. Yin X. Lill M.A. Danielson M.L. Freiser H. Huang J. Long-chain carboxychromanols, metabolites of vitamin E, are potent inhibitors of cyclooxygenases.Proc. Natl. Acad. Sci. USA. 2008; 105: 20464-20469Crossref PubMed Scopus (147) Google Scholar). from δT 5-lipoxygenase activity and leukotriene in stimulated On the other hand, tocopherols have on 5-lipoxygenase activity, and their of leukotriene in cells with Z. Yin X. Jiang Q. Natural forms of vitamin E and 13′-carboxychromanol, a long-chain vitamin E metabolite, inhibit leukotriene generation from stimulated neutrophils by blocking calcium influx and suppressing 5-lipoxygenase activity, respectively.J. Immunol. 2011; 186: 1173-1179Crossref PubMed Scopus Google Scholar). In 13′-COOHs apoptosis in liver cells more than M. D. S. S. D. M. M. effects of long-chain vitamin E metabolites in HepG2 cells are by Radic. Biol. Med. PubMed Scopus Google Scholar). 13′-OH and from αT are found to have activities M. A. M. F. M. S. long-chain metabolite the response of Nutr. Res. PubMed Scopus Google Scholar). these of long-chain metabolites, is to that these compounds a role in vitamin E-mediated effects in (1Jiang Q. Natural forms of vitamin E: metabolism, antioxidant, and anti-inflammatory activities and their role in disease prevention and therapy.Free Radic. Biol. Med. 2014; 72: 76-90Crossref PubMed Scopus (518) Google which further In we have an LC/MS/MS assay that carboxychromanols, and sulfated vitamin E metabolites. This be useful for further of of vitamin E metabolite and their bioavailability in tissues and as as for of potential of metabolites to vitamin E-mediated effects in animals and 5′-, 7′-, 9′-, 11′-, and 13′-carboxychromanol sulfated 9′-, 11′-, and 13′-carboxychromanol 13′-hydroxychromanol α-, β-, or δ-tocopherol α-, β-, or sulfated 3′-COOH