Identification and quantitation of novel vitamin E metabolites, sulfated long-chain carboxychromanols, in human A549 cells and in rats

Abstract

The metabolism of vitamin E involves oxidation of the phytyl chain to generate the terminal metabolite 7,8-dimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (CEHC) via intermediate formation of 13'-hydroxychromanol and long-chain carboxychromanols. Conjugated (including sulfated) metabolites were reported previously but were limited to CEHCs. Here, using electrospray and inductively coupled plasma mass spectrometry, we discovered that gamma-tocopherol (gamma-T) and delta-T were metabolized to sulfated 9'-, 11'-, and 13'-carboxychromanol (9'S, 11'S, and 13'S) in human A549 cells. To further study the metabolites, we developed a HPLC assay with fluorescence detection that simultaneously analyzes sulfated and nonconjugated intermediate metabolites. Using this assay, we found that sulfated metabolites were converted to nonconjugated carboxychromanols by sulfatase digestion. In cultured cells, approximately 45% long-chain carboxychromanols from gamma-T but only 10% from delta-T were sulfated. Upon supplementation with gamma-T, rats had increased tissue levels of 9'S, 11'S, and 13'S, 13'-hydroxychromanol, 13'-carboxychromanol, and gamma-CEHC. The plasma concentrations of combined sulfated long-chain metabolites were comparable to or exceeded those of CEHCs and increased proportionally with the supplement dosages of gamma-T. Our study identifies sulfated long-chain carboxychromanols as novel vitamin E metabolites and provides evidence that sulfation may occur parallel with beta-oxidation. In addition, the HPLC fluorescence assay is a useful tool for the investigation of vitamin E metabolism.

Fig. 1

Identification of metabolites from γ-tocopherol (γ-T) in cultured A549 cells. A: HPLC (with fluorescence detection) of γ-T metabolites accumulated in the cell culture media after A549 cells were incubated with 50 μM γ-T for 72 h. 9′, 11′, and 13′ represent 9′-, 11′-, and 13′-carboxychromanol (COOH), and 9′S, 11′S, and 13′S stand for sulfated 9′-, 11′-, and 13′-carboxychromanol, respectively. B, C: Electrospray ionization mass spectrometry of 13′-COOH and 13′S, respectively.

Fig. 2

A: Inductively coupled plasma mass spectrometry (ICP-MS) detection of ³⁴S for isolated 9′S. Traces 1, 2, and 3 show ³⁴S intensity of 2% HNO₃ (the solvent blank), an isolated mobile phase control, and isolated 9′S, respectively. B: HPLC of the isolated 9′S, which was incubated in 0.1 M sodium acetate (pH 5) at 37°C for 1 h (upper trace), and after 9′S was digested with a sulfatase under the same conditions (lower trace). C: Relative responses of metabolites to electrochemical oxidation (lower trace) compared with fluorescent signal detection (upper trace). Metabolites from γ-T extracted from cell culture media were separated by HPLC and detected by fluorescence detection (excitation and emission wavelengths were set at 292 and 327 nm, respectively), followed by coulometric detection at 400 mV using a model 5011 analytical cell.

Fig. 3

A linear relationship exists between the mobile phase composition (percentage solvent B) and the fluorescence response (peak area). Twenty microliters of γ-tocotrienol (1 μM) was injected onto the HPLC apparatus under isocratic conditions with 100, 90, 85, and 80% solvent B and the corresponding 0, 10, 15, and 20% solvent A, respectively. The fluorescence responses under these various conditions were plotted with the corresponding mobile phase content, which showed a linear relationship and can be expressed by the equation y = 0.187x − 6.34 (R² = 0.994), where x is the percentage of solvent B and y is the area (×10⁻⁶) of 1 μM of the standard.

Fig. 4

Time-dependent accumulation of various metabolites in cultured cells. Ten micromolar γ-T (A) and δ-T (B) were incubated in confluent A549 cells for 24, 48, or 72 h, and media were collected. Metabolites were quantified using HPLC assay with fluorescence detection. Data shown are averages ± SD of three independent experiments.

Fig. 5

Metabolites identified in rat plasma upon γ-T supplementation. Wistar rats were fed a single dose of γ-T at 90 mg/kg in corn oil (upper trace) or tocopherol-stripped corn oil alone (lower trace). Six hours later, rats were euthanized and plasma as well as other tissues were collected. CEHC, 7,8-dimethyl-2-(β-carboxyethyl)-6-hydroxychroman; 13′-OH, 13′-hydroxychromanol; 13′, 13′-carboxychromanol; 9′S, 11′S, 13′S, sulfated 9′-, 11′-, and 13′-carboxychromanol.

Scheme 1

Structures of tocopherols and their terminal metabolite, 7,8-dimethyl-2-(β-carboxyethyl)-6-hydroxychroman (CEHC). α-T, β-T, γ-T, or δ-T, α-, β-, γ-, or δ-tocopherol.

Scheme 2

Proposed metabolism of vitamin E forms based on the identified metabolites. Based on previous studies, vitamin E forms are metabolized via ω-hydroxylation and β-oxidation. The present study suggests that sulfation is a parallel process to β-oxidation. Dashed arrows indicate hypothesized reaction pathways that need to be further characterized.