Methods for efficient analysis of tocopherols, tocotrienols and their metabolites in animal samples with HPLC-EC

Abstract

Tocopherols and tocotrienols, collectively known as vitamin E, have received a great deal of attention because of their interesting biological activities. In the present study, we reexamined and improved previous methods of sample preparation and the conditions of high-performance liquid chromatography for more accurate quantification of tocopherols, tocotrienols and their major chain-degradation metabolites. For the analysis of serum tocopherols/tocotrienols, we reconfirmed our method of mixing serum with ethanol followed by hexane extraction. For the analysis of tissue samples, we improved our methods by extracting tocopherols/tocotrienols directly from tissue homogenate with hexane. For the analysis of total amounts (conjugated and unconjugated forms) of side-chain degradation metabolites, the samples need to be deconjugated by incubating with β-glucuronidase and sulfatase; serum samples can be directly used for the incubation, whereas for tissue homogenates a pre-deproteination step is needed. The present methods are sensitive, convenient and are suitable for the determination of different forms of vitamin E and their metabolites in animal and human studies. Results from the analysis of serum, liver, kidney, lung and urine samples from mice that had been treated with mixtures of tocotrienols and tocopherols are presented as examples.

Keywords: Metabolites; Mice; Tissue levels; Tocopherols; Tocotrienols.

Fig. 1

Structures of γ-tocopherol and γ-tocotrienol and their side-chain degradation pathways. The side-chain degradation is initiated by ω-oxidation and followed by cycles of β-oxidation, each reducing the chain length by two carbons. The conversion of γ-CMHenHC to CMBHC has been shown to be catalyzed by the presence of auxiliary enzymes 2,4-dienoyl-CoA reductase and 3,2-enoyl-CoA isomerase [8]. The structures and degradation of other forms of tocopherols and tocotrienols are similar, as the chromanol ring is trimethylated at the 5-, 7- and 8-positions in α-T/T3, and methylated at the 8-position in δ-T/T3.

Fig. 2

HPLC chromatograms of tocopherols and tocotrienols (A) and CEHCs and CMBHCs (B) in mouse serum, lung, and liver. All samples were collected at 3 h after the fourth oral dose of tocotrienol mixture. Solid lines, samples from mice treated with 4 daily doses of m-T3; dotted lines, samples from mice treated with γ-TmT-enriched diet for 18 days.

Fig. 3

Calibration curves for tocopherols and tocotrienols (A) and CEHCs and CMBHCs (B). The injection volume was 50 μl. Each data point represents the average of two measurements.

Fig. 4

Long-chain metabolites determined in mouse tissues together with their parent tocopherols. The tissue samples were from mice treated with a γ-TmT-supplemented diet for 18 days. For the kidney and lung samples, “↓” was added to identify the peaks.

Fig. 5

Serum levels of tocopherols and tocotrienols (A) and CEHCs and CMBHCs (B): Samples were collected at 3, 7, 24, 27, 48, 51, 72, and 75 h in the experiment with daily oral dose of tocotrienols (m-T3). The data are expressed as mean ± SD (n = 6).