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

Abstract

To evaluate vitamin E metabolism, a method was developed to quantitate liver α- and γ-tocopherol metabolites, α-carboxyethyl hydroxychroman [α-CEHC; 2,5,7,8-tetramethyl-2-(2′-carboxyethyl)-6-hydroxychroman] and γ-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 α- and γ-CEHCs added to liver homogenates ranged from 77% to 87%. Detection limits of α- and γ-CEHC were 20 fmol, with a linear detector response from 0.025 to 20 pmol injected. Corresponding with an increase in liver α-tocopherol, the MS peak for liver α-CEHC (mass-to-charge ratio 277.8) increased 80-fold (0.18 ± 0.01 to 15 ± 2 nmol/g). Liver α-CEHC concentrations were correlated with serum α-CEHC, liver α-tocopherol, and serum α-tocopherol (P < 0.001 for each comparison). α-CEHC represented 0.5–1% of the liver α-tocopherol concentration.Thus, LC-MS can be successfully used to quantitate α- and γ-CEHC in liver samples. These data suggest that in times of excess liver α-tocopherol, increased metabolism of α-tocopherol to α-CEHC occurs.

Keywords

• α-carboxyethyl hydroxychroman
• γ-carboxyethyl hydroxychroman
• mass spectrometry
• vitamin E metabolism
• α-tocopherol
• γ-tocopherol