Measurement of the Vitamin E Metabolites, Carboxyethyl Hydroxychromans (CEHCs), in Biological Samples

Scott W. Leonard1, Maret G. Traber1

1 Linus Pauling Institute, Oregon State University, Corvallis, Oregon
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 7.8
DOI:  10.1002/0471140856.tx0708s29
Online Posting Date:  September, 2006
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Metabolites of α‐ and γ‐tocopherol, 2,5,7,8‐tetramethyl‐2‐(2′‐carboxyethyl)‐6‐hydroxychroman (α‐CEHC) and 2,7,8‐trimethyl‐2‐(β‐carboxyethyl)‐6‐hydroxychroman (γ‐CEHC), respectively, are produced in the liver and have been measured in biological fluids and tissue. Compared to α‐tocopherol concentrations, metabolite concentrations are as much as a factor of a thousand lower, requiring extremely sensitive methodology to attain accurate measurements. This unit presents a protocol for CEHC extraction from biological samples, and describes very specific and sensitive HPLC/MS analysis.

Keywords: α‐CEHC; γ‐CEHC; mass spectrometry; vitamin E metabolism; α‐tocopherol; γ‐tocopherol

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Table of Contents

  • Basic Protocol 1: HPLC‐Mass Spectrometric Analysis of α‐ and γ‐CEHCs
  • Collection and Storage Procedures for Biological Samples Intended for CEHC Analysis
  • Support Protocol 1: Preparation of Plasma Samples
  • Support Protocol 2: Preparation of Urine Samples
  • Support Protocol 3: Preparation of Tissue Samples
  • Support Protocol 4: Quantification of 24‐Hr Urinary Creatine
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: HPLC‐Mass Spectrometric Analysis of α‐ and γ‐CEHCs

  • Mobile phase solutions A and B (see recipe)
  • Plasma (see protocol 2), urine (see protocol 3), or tissue homogenate (see protocol 4)
  • 1% (w/v) ascorbic acid solution (see recipe)
  • Trolox solution (see recipe)
  • 100% ethanol
  • β‐glucuronidase solution (300,000 U/g β‐glucuronidase activity and 10,000 U/g sulfatase activity, Sigma‐Aldrich type H‐1 or equivalent; see recipe)
  • 12 M hydrochloric acid (HCl; muriatic acid)
  • Diethyl ether (ethyl ether anhydrous, Mallinekrodt Baker)
  • 50% (v/v) methanol solution (see recipe)
  • Phosphate buffered saline (PBS)/0.05% (w/v) EDTA solution (see recipe)
  • Standard mixture of γ‐CEHC (Cayman Chemical) and α‐CEHC (currently not commercially available, can be prepared as described in Mazzini et al., ; see recipe for working standard; 3.78 µM γ‐CEHC and 1.80 µM α‐CEHC final)
  • Ascorbic acid (USP grade)
  • Acetic acid, glacial (certified A.C.S.)
  • High‐performance liquid chromatography (HPLC) system, including:
    • Gradient pump system (e.g., Waters Alliance 2690 separations module or equivalent)
    • Vacuum degasser, optional
    • Column oven, 30°C
    • Cooled autosampler, 50 µl injection loop, 4°C
    • Mass spectrometer (Waters Micromass ZQ 2000 single quadrupole, or equivalent) and accessories, including: Waters Masslynx version 4.0 data acquisition and integration package and electrospray ionization probe
    • HPLC column: 15‐cm × 3.0–mm (i.d.) SymmetryShield RP18 3.5‐µm particle size (Waters)
    • Guard column: 2‐cm × 3.9–mm (i.d.) SymmetryShield Sentry RP18 3.5‐µm particle size (Waters)
  • 16 × 100–mm screw‐capped, disposable, borosilicate‐glass test tubes
  • 37°C incubator (e.g., Isotemp, Fisher Scientific)
  • Glass pipets
  • 13 × 100–mm disposable, borosilicate glass test tube
  • N‐EVAP nitrogen evaporation system (12 position; Organomation Associates, Inc.)
  • Low‐volume HPLC injection vials with caps (e.g., SUN SRi)
  • Variable‐speed tissue homogenizer (1000 to 27,000 rpm; e.g., Polytron, Brinkmann) with a 7‐mm foam reducing generator (Brinkmann)
  • 1.5‐ml microcentrifuge tubes

Support Protocol 1: Preparation of Plasma Samples

  • Anticoagulated blood sample (blood that has been collected into a Vacutainer containing EDTA or heparin)
  • Liquid nitrogen
  • Centrifuge (e.g., Beckman TJ‐R or equivalent), 4°C
  • 2‐ml cryogenic storage vials (Nalgene)

Support Protocol 2: Preparation of Urine Samples

  • 24‐hr urine collection
  • 24‐hr urine collection container (3‐liter, VWR)
  • 4‐liter polypropylene graduated cylinder (Nalgene)
  • 2‐ml cryogenic storage vials (Nalgene)

Support Protocol 3: Preparation of Tissue Samples

  • Isolated intact organ
  • Phosphate‐buffered saline (PBS)
  • Liquid nitrogen
  • Aluminum foil

Support Protocol 4: Quantification of 24‐Hr Urinary Creatine

  • Frozen urine sample (see protocol 3)
  • Creatinine assay kit (Cayman Chemical or equivalent)
  • Spectrophotometer reading at 500 nm (Beckman DU 640 or equivalent)
  • 1‐ml polystyrene cuvettes
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Literature Cited

   Birringer, M., Drogan, D., and Brigelius‐Flohe, R. 2001. Tocopherols are metabolized in HepG2 cells by side chain omega‐oxidation and consecutive beta‐oxidation. Free Radic. Biol. Med. 31:226‐232.
   Birringer, M., Pfluger, P., Kluth, D., Landes, N., and Brigelius‐Flohe, R. 2002. Identities and differences in the metabolism of tocotrienols and tocopherols in HepG2 cells. J. Nutr. 132:3113‐3118.
   Brigelius‐Flohe, R. 2003. Vitamin E and drug metabolism. Biochem. Biophys. Res. Commun. 305:737‐740.
   Brigelius‐Flohé, R. and Traber, M.G. 1999. Vitamin E: Function and metabolism. FASEB J. 13:1145‐1155.
   Brigelius‐Flohe, R., Roob, J.M., Tiran, B., Wuga, S., Ribalta, J., Rock, E., and Winklhofer‐Roob, B.M. 2004. The effect of age on vitamin E status, metabolism, and function: Metabolism as assessed by labeled tocopherols. Ann. N. Y. Acad. Sci. 1031:40‐43.
   Bruno, R.S., Leonard, S.W., Li, J., Bray, T.M., and Traber, M.G. 2005. Lower plasma α‐carboxyethyl‐hydroxychroman after deuterium labeled α‐tocopherol supplementation suggests decreased vitamin E metabolism in smokers. Am. J. Clin. Nutr. 81:1052‐1059.
   Galli, F., Lee, R., Dunster, C., and Kelly, F.J. 2002. Gas chromatography mass spectrometry analysis of carboxyethyl‐hydroxychroman metabolites of alpha‐ and gamma‐tocopherol in human plasma. Free Radic. Biol. Med. 32:333‐340.
   Himmelfarb, J., Kane, J., McMonagle, E., Zaltas, E., Bobzin, S., Boddupalli, S., Phinney, S., and Miller, G. 2003. Alpha and gamma tocopherol metabolism in healthy subjects and patients with end‐stage renal disease. Kidney Int. 64:978‐991.
   Kantoci, D., Wechter, W.J., Murray, E.D.J., Dewind, S.A., Borchardt, D., and Khan, S.I. 1997. Endogenous natriuretic factors 6: The stereochemistry of a natriuretic gamma‐tocopherol metabolite LLU‐alpha. J. Pharmacol. Exp. Ther. 282:648‐656.
   Kliewer, S.A. 2003. The nuclear pregnane x receptor regulates xenobiotic detoxification. J. Nutr. 133:2444S‐2447S.
   Kluth, D., Landes, N., Pfluger, P., Muller‐Schmehl, K., Weiss, K., Bumke‐Vogt, C., Ristow, M., and Brigelius‐Flohe, R. 2005. Modulation of Cyp3a11 mRNA expression by alpha‐tocopherol but not gamma‐tocotrienol in mice. Free Radic. Biol. Med. 38:507‐514.
   Leonard, S.W., Gumpricht, E., Devereaux, M.W., Sokol, R.J., and Traber, M.G. 2005a. Quantitation of rat liver vitamin E metabolites by LC‐MS during high‐dose vitamin E administration. J. Lipid Res. 46:1068‐1075.
   Leonard, S.W., Paterson, E., Atkinson, E.J., Ramakrishnan, R., Cross, C.E., and Traber, M.G. 2005b. Studies in humans using deuterium‐labeled α‐ and γ‐tocopherol demonstrate faster plasma γ‐tocopherol disappearance and greater γ‐metabolite production. Free Radic. Biol. Med. 38:857‐866.
   Lodge, J.K., Traber, M.G., Elsner, A., and Brigelius‐Flohe, R. 2000. A rapid method for the extraction and determination of vitamin E metabolites in human urine. J. Lipid Res. 41:148‐154.
   Lodge, J.K., Ridlington, J., Vaule, H., Leonard, S.W., and Traber, M.G. 2001. α‐ and γ‐Tocotrienols are metabolized to carboxyethyl‐hydroxychroman (CEHC) derivatives and excreted in human urine. Lipids 36:43‐48.
   Mazzini, F., Netscher, T., and Salvadori, P. 2004. First synthesis of rac‐(5‐2H3)‐alpha‐CEHC, a labeled analogue of a major vitamin E metabolite. J. Org. Chem. 69:9303‐9306.
   Pope, S.A., Clayton, P.T., and Muller, D.P. 2000. A new method for the analysis of urinary vitamin E metabolites and the tentative identification of a novel group of compounds. Arch. Biochem. Biophys. 381:8‐15.
   Pope, S.A., Burtin, G.E., Clayton, P.T., Madge, D.J., and Muller, D.P. 2001. New synthesis of (±)‐alpha‐CMBHC and its confirmation as a metabolite of alpha‐tocopherol (vitamin E). Bioorg. Med. Chem. 9:1337‐1343.
   Pope, S.A., Burtin, G.E., Clayton, P.T., Madge, D.J., and Muller, D.P. 2002. Synthesis and analysis of conjugates of the major vitamin E metabolite, alpha‐CEHC. Free Radic. Biol. Med. 33:807‐817.
   Schuelke, M., Elsner, A., Finckh, B., Kohlschutter, A., Hubner, C., and Brigelius‐Flohe, R. 2000. Urinary alpha‐tocopherol metabolites in alpha‐tocopherol transfer protein‐deficient patients. J. Lipid Res. 41:1543‐1551.
   Schultz, M., Leist, M., Petrzika, M., Gassmann, B., and Brigelius‐Flohé, R. 1995. 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. 62:1527S‐1534S.
   Schultz, M., Leist, M., Elsner, A., and Brigelius‐Flohe, R. 1997. Alpha‐carboxyethyl‐6‐hydroxychroman as urinary metabolite of vitamin E. Methods Enzymol. 282:297‐310.
   Simon, E.J., Eisengart, A., Sundheim, L., and Milhorat, A.T. 1956. The metabolism of vitamin E. II. Purification and characterization of urinary metabolites of alpha‐tocopherol. J. Biol. Chem. 221:807‐817.
   Sontag, T.J. and Parker, R.S. 2002. Cytochrome P450 omega‐hydroxylase pathway of tocopherol catabolism: Novel mechanism of regulation of vitamin E status. J. Biol. Chem. 277:25290‐25296.
   Stahl, W., Graf, P., Brigelius‐Flohe, R., Wechter, W., and Sies, H. 1999. 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. 275:254‐259.
   Swanson, J.E., Ben, R.N., Burton, G.W., and Parker, R.S. 1999. 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. 40:665‐671.
   Traber, M.G. 2004. Vitamin E, nuclear receptors and xenobiotic metabolism. Arch. Biochem. Biophys. 423:6‐11.
   Traber, M.G., Elsner, A., and Brigelius‐Flohe, R. 1998. Synthetic as compared with natural vitamin E is preferentially excreted as alpha‐CEHC in human urine: Studies using deuterated alpha‐tocopheryl acetates. FEBS Lett. 437:145‐148.
   Traber, M.G., Siddens, L.K., Leonard, S.W., Schock, B., Gohil, K., Krueger, S.K., Cross, C.E., and Williams, D.E. 2005. α‐Tocopherol modulates Cyp3a expression, increases γ‐CEHC production and limits tissue γ‐tocopherol accumulation in mice fed high γ‐tocopherol diets. Free Radic. Biol. Med. 38:773‐785.
   Wechter, W.J., Kantoci, D., Murray, E.D.J., D'Amico, D.C., Jung, M.E., and Wang, W.H. 1996. A new endogenous natriuretic factor: LLU‐alpha. Proc. Natl. Acad. Sci. U.S.A. 93:6002‐6007.
   You, C.S., Sontag, T.J., Swanson, J.E., and Parker, R.S. 2005. Long‐chain carboxychromanols are the major metabolites of tocopherols and tocotrienols in A549 lung epithelial cells but not HepG2 cells. J. Nutr. 135:227‐232.
Key References
   Leonard et al., 2005a. See above.
  One of the methods on which the is based and original report on tissue CEHC measurement in tissue homogenates.
   Lodge et al., 2000. See above.
  One of the methods on which the is based.
   Sontag and Parker, 2002. See above.
  Identification of vitamin E intermediary metabolites, including CEHC.
   Birrenger et al., 2002. See above.
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