Original contributionSynthesis and analysis of conjugates of the major vitamin E metabolite, α-CEHC
Introduction
Vitamin E is a generic term for the tocopherols and tocotrienols, which have saturated and unsaturated side chains, respectively. Each group can occur in α, β, γ, and δ-forms, which differ in the number and position of methyl groups on the chroman ring with α-tocopherol being the most abundant form of vitamin E in vivo.
The biological activity of α-tocopherol has been attributed principally to its ability to act as an antioxidant. It is the major lipid soluble antioxidant in vivo [1] and protects lipids from peroxidation. More recently non-antioxidant roles of α-tocopherol have also been demonstrated. α-Tocopherol, in contrast to structurally related antioxidants such as β-tocopherol, is able to inhibit protein kinase C (PKC) activity by changing its phosphorylation state [2]. Other non-antioxidant roles of α-tocopherol include inhibition of vascular smooth muscle cell proliferation [3] as well as upregulation of the expression of genes for α-tropomyosin [4] and connective tissue growth factor [5].
A study of the metabolism of vitamin E could provide useful insights into the function(s) of vitamin E in vivo as it might be expected to differ in health and disease depending on the body’s requirement and its specific roles at that time. The monitoring of vitamin E metabolites may therefore be useful in studying disease progression and the effects of interventions (such as antioxidant supplements or drugs), as well as giving clues to the specific role(s) of vitamin E in vivo.
Most work studying vitamin E metabolism has concentrated on the metabolites of α-tocopherol since it accounts for over 90% of vitamin E in vivo. Two groups of metabolites have been identified (Fig. 1). The first group comprises α-CEHC (1; see “Experimental Procedures” for IUPAC names) and α-carboxymethylbutyl-hydroxychroman (α-CMBHC; 2), which are formed by metabolism of the phytyl side chain and are thought to be products of excess/adequate vitamin E [6], [7], [8], [9]. The second group is made up of α-tocopheronic acid (3) and its γ-lactone, α-tocopheronolactone (4), which have an oxidized chroman ring and are considered to be metabolites produced after α-tocopherol has reacted with oxidants [10], [11]. α-CEHC (1) is the major metabolite of α-tocopherol and it is unclear whether the small amounts of α-tocopheronolactone (4) observed are real or produced artifactually by oxidation of α-CEHC (1) during the methodological work up [6], [8]. Confirmation of the authenticity of α-tocopheronolactone (4) is important, because it could be potentially useful as a biomarker of oxidative stress.
Indirect evidence using β-glucuronidase and sulphatase enzymes has indicated that vitamin E metabolites are excreted in the urine as highly polar sulphate or glucuronide conjugates [12], [13], [14]. The high polarity of these conjugates makes analysis with common techniques such as high performance liquid chromatography (HPLC) or gas chromatography/mass spectrometry (GC-MS) difficult. So far every reported method for the analysis of urinary vitamin E metabolites has analyzed the metabolites after acid or enzymatic deconjugation, thus simplifying the separation and characterization of the metabolites by GC-MS or HPLC. However, there are a number of potential problems with these methods. Both acid and enzymatic deconjugation can lead to oxidation/degradation of the metabolites owing to their harshness or length, respectively. Indeed, as noted above, α-CEHC (1) has been shown to oxidatively convert to α-tocopheronolactone (4) under mildly oxidizing conditions [6]. Even with careful handling, conversion occurs, thus making measurement of α-tocopheronolactone (4), the minor component, unreliable. Other potential problems with these methods include incomplete hydrolysis of the conjugated metabolites, the length of the protocol, the number of handling steps, and the need for derivatization of the unconjugated metabolites prior to analysis by GC-MS. Immediate improvements in terms of reliability and time could be made if methods to analyze the intact conjugates were developed.
Tandem mass-spectrometry using an electrospray or fast atom bombardment (FAB) source has been used for the analysis of a variety of conjugated metabolites. Preliminary data, described here, indicates that tandem mass spectrometry can also be used for the analysis of urinary vitamin E metabolites. However, in order to fully characterize and quantitate conjugated urinary vitamin E metabolites, chemical standards of the conjugated metabolites are required.
Here we describe the first direct analysis of conjugated vitamin E metabolites using tandem mass spectrometry and also describe the first synthesis of the glucuronide and sulphate conjugates of the major α-tocopherol metabolite, α-CEHC (1).
Section snippets
Materials and methods
All chemicals were obtained from Sigma-Aldrich Chemical Company (St. Louis, MO, USA) unless otherwise stated. Solvents and reagents were used without further purification except tetrahydrofuran (THF) which was dried over sodium. Reactions were monitored by thin layer chromatography (TLC) on precoated silica gel plates (Kieselgel 60 F254, Merck Ltd.). Purification was performed by flash chromatography using silica gel (particle size 40–63 μM, Merck Ltd.). 1H and 13C NMR spectra were recorded on
Analysis of conjugated urinary vitamin E metabolites in scan mode
In order to assess the ability of electrospray tandem mass spectrometry to analyze conjugated vitamin E metabolites and to obtain preliminary data on the type of conjugates present, urine samples from subjects given oral doses of RRR-α-tocopheryl acetate (1000 mg/d for 3 weeks) were analyzed. The mass spectrometer was initially operated in negative ion scan mode (without using the collision cell) in order to identify peaks corresponding to possible conjugated vitamin E metabolites.
Peaks at m/z
Discussion
Here we have described for the first time the synthesis and analysis of conjugated vitamin E metabolites. After α-tocopherol supplementation, a peak at m/z 453 was observed, which was characterized as α-CEHC glucuronide (8) by comparison of its CID spectrum with that of an authentic standard. A minor peak in urine, having a mass consistent with α-CEHC sulphate (12) (m/z 357) and displaying a characteristic fragment of sulphate conjugates (m/z 80), could not be unambiguously characterized using
Acknowledgements
We thank the Engineering and Physical Science Research Council and the Wellcome Trust for financial support.
References (19)
- et al.
Is vitamin E the only lipid-soluble antioxidant in human blood plasma and erythrocyte membranes?
Arch. Biochem. Biophys.
(1983) - et al.
Modulation of alpha-tropomyosin expression by alpha-tocopherol in rat vascular smooth muscle cells
FEBS Lett.
(1999) - et al.
Non-antioxidant functions of alpha-tocopherol in smooth muscle cells
J. Nutr.
(2001) - et al.
Synthetic as compared with natural vitamin E is preferentially excreted as alpha-CEHC in human urinestudies using deuterated alpha-tocopheryl acetates
FEBS Lett.
(1998) - et al.
A new method for the analysis of urinary vitamin E metabolites and the tentative identification of a novel group of compounds
Arch. Biochem. Biophys.
(2000) - et al.
Urinary alpha-tocopherol metabolites in alpha-tocopherol transfer protein-deficient patients
J. Lipid Res.
(2000) - et al.
The absorption and excretion of D-α-tocopheryl-5-methyl-C14-succinate
J. Biol. Chem.
(1956) - et al.
Purification and characterization of urinary metabolites of α-tocopherol
J. Biol. Chem.
(1956) - et al.
Novel urinary metabolite of D-delta-tocopherol in rats
J. Lipid Res.
(1984)