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Journal of Lipid Research, Vol. 46, 1068-1075, May 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Methods

Quantitation of rat liver vitamin E metabolites by LC-MS during high-dose vitamin E administration

Scott W. Leonard^*, Eric Gumpricht, Michael W. Devereaux, Ronald J. Sokol and Maret G. Traber^1,*

^* Linus Pauling Institute, Oregon State University, Corvallis, OR
Section of Pediatric Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, University of Colorado Health Sciences Center and The Children's Hospital, Denver, CO

¹ To whom correspondence should be addressed. e-mail: maret.traber{at}oregonstate.edu

To evaluate vitamin E metabolism, a method was developed to quantitate liver - and -tocopherol metabolites, -carboxyethyl hydroxychroman [-CEHC; 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman] and -CEHC [2,7,8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman], respectively. Vitamin E supraenriched livers were obtained from rats that were injected with vitamin E daily for 18 days. Liver samples (50 mg) were homogenized, homogenate CEHC-conjugates were hydrolyzed, CEHCs were extracted with ethyl ether, and then CEHCs were quantitated using liquid chromatography-mass spectrometry (LC-MS). Precision, based on intersample variability, ranged from 1% to 3%. Recovery of - and -CEHCs added to liver homogenates ranged from 77% to 87%. Detection limits of - and -CEHC were 20 fmol, with a linear detector response from 0.025 to 20 pmol injected. Corresponding with an increase in liver -tocopherol, the MS peak for liver -CEHC (mass-to-charge ratio 277.8) increased 80-fold (0.18 ± 0.01 to 15 ± 2 nmol/g). Liver -CEHC concentrations were correlated with serum -CEHC, liver -tocopherol, and serum -tocopherol (P < 0.001 for each comparison). -CEHC represented 0.5–1% of the liver -tocopherol concentration.

Thus, LC-MS can be successfully used to quantitate - and -CEHC in liver samples. These data suggest that in times of excess liver -tocopherol, increased metabolism of -tocopherol to -CEHC occurs.

Abbreviations: CEHC, carboxyethyl hydroxychroman; LC-MS, liquid chromatography-mass spectrometry; MDR1, multidrug resistance protein-1; m/z, mass-to-charge ratio; SXR, steroid and xenobiotic receptor; UGT1A1, UDP-glucuronosyltransferase-1A1

Supplementary key words -carboxyethyl hydroxychroman • -carboxyethyl hydroxychroman • mass spectrometry • vitamin E metabolism • -tocopherol • -tocopherol • liquid chromatography-mass spectrometry

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