Analysis of multiple metabolites of tocopherols and tocotrienols in mice and humans

Abstract

Tocopherols and tocotrienols, collectively known as vitamin E, are essential antioxidant nutrients. The biological fates and metabolite profiles of the different forms are not clearly understood. The objective of this study is to simultaneously analyze the metabolites of different tocopherols and tocotrienols in mouse and human samples. Using HPLC/electrochemical detection and mass spectrometry, 18 tocopherol-derived and 24 tocotrienol-derived side-chain degradation metabolites were identified in fecal samples. Short-chain degradation metabolites, in particular gamma- and delta-carboxyethyl hydroxychromans (CEHCs) and carboxymethylbutyl hydroxychromans (CMBHCs) were detected in urine, serum, and liver samples, with tocopherols additionally detected in serum and liver samples. The metabolite profiles of tocotrienols and tocopherols were similar, but new tocotrienol metabolites with double bonds were identified. This is the first comprehensive report describing simultaneous analysis of different side-chain metabolites of tocopherols and tocotrienols in mice and humans. Urinary metabolites may serve as useful biomarkers for the nutritional assessment of vitamin E.

Figure 1

Structures of tocopherols/tocotrienols and the proposed metabolic pathways. The structures are illustrated with γ-tocopherol and γ-tocotrienol, which are both dimethylated at the 7- and 8- positions. The chromanol ring is trimethylated at 5-, 7-, and 8-positions in α-tocopherol, dimethylated at 5- and 8-positions in β-tocopherol, and methylated at 8-position in δ-tocopherol. The arrows (from right to left) indicate the sequential side chain degradation and the resulting metabolites.

Figure 2

Identification of multiple tocopherol/tocotrienol metabolites in mouse fecal samples. A: HPLC chromatogram of tocopherol/tocotrienol metabolites. The peaks eluted between 13.5 – 30 min for the tocotrienol group were not assigned. They could be derived from tocotrienols or other compounds. B: Representative MS² spectra and structural elucidation of deprotonated ions from γ-derived metabolites.

Figure 3

HPLC chromatogram of tocopherol metabolites in human fecal samples. Fecal samples were collected at 0, 12, 24, and 48 h after ingestion of γ-tocopherol-rich tocopherol softgels. The responses of tocopherols (right) and tocopherol metabolites (left) are shown in different scales.

Figure 4

HPLC chromatograms of tocopherol/tocotrienol metabolites in urine samples. A: tocopherol/tocotrienol metabolites in mouse urine samples. B: tocopherol metabolites in enzyme-hydrolyzed and un-hydrolyzed human urine samples collected at 0, 12, 24, and 48 h. C: tocopherol metabolites in pre-dose human urine samples after and before enzymatic hydrolysis shown in a more sensitive scale.

Figure 5

HPLC chromatogram of tocopherol metabolites in mouse (M) and human (H) serum samples as well as mouse liver samples. The responses of tocopherols (right) and tocopherol metabolites (left) are shown in different scales.