Associations of metabolomic profiles with circulating vitamin E and urinary vitamin E metabolites in middle-aged individuals.

Vitamin E (α-tocopherol [α-TOH]) is transported in lipoprotein particles in blood, but little is known about the transportation of its oxidized metabolites. In the Netherlands Epidemiology of Obesity Study, we aimed to investigate the associations of 147 circulating metabolomic measures obtained through targeted nuclear magnetic resonance with serum α-TOH and its urinary enzymatic (α-CEHC) and oxidized (α-TLHQ) metabolites from 24-h urine quantified by liquid chromatography with tandem mass spectrometry. Multivariable linear regression analyses, in which multiple testing was taken into account, were performed to assess associations between metabolomic measures (determinants; standardized to mean = 0, SD = 1) and vitamin E metabolites (outcomes), adjusted for demographic factors. We analyzed 474 individuals (55% women, 45% men) with a mean (SD) age of 55.7 (6.0) y. Out of 147 metabolomic measures, 106 were associated (P < 1.34 × 10-3) with serum α-TOH (median ß [interquartile range] = 0.416 [0.383-0.466]), predominantly lipoproteins associated with higher α-TOH. The associations of metabolomic measures with urinary α-CEHC have directions similar to those with α-TOH, but effect sizes were smaller and non-significant (median ß [interquartile range] = 0.065 [0.047-0.084]). However, associations of metabolomic measures with urinary α-TLHQ were markedly different from those with both serum α-TOH and urinary α-CEHC, with negative and small-to-null relations to most very-low-density lipoproteins and amino acids. Therefore, our results highlight the differences in the lipoproteins involved in the transportation of circulating α-TOH and oxidized vitamin E metabolites. This indicates that circulating α-TOH may be representative of the enzymatic but not the antioxidative function of vitamin E.

Daily Consumption of Oregon Hazelnuts Affects α-Tocopherol Status in Healthy Older Adults: A Pre-Post Intervention Study.

Background: Inadequate vitamin E and magnesium intakes are of concern for older adults owing to the associated incidence of age-related diseases. Objective: This study was designed to determine the extent to which a 16-wk intervention with hazelnuts alters vitamin E and magnesium status in a group of older men and women, and used a pre-post intervention design without a control group to adjust for temporal changes. Methods: Participants (n = 32 including 22 women; mean ± SD age: 63 ± 6 y) consumed hazelnuts (∼57 g/d) for 16 wk. Blood and urine samples and anthropomorphic measures were taken at the start and end of the intervention to determine plasma concentrations of α-tocopherol and serum concentrations of magnesium, lipids, glucose, insulin, and high-sensitivity C-reactive protein along with urinary vitamin E metabolites; several other micronutrients were measured by a lymphocyte proliferation assay. There were 3 primary endpoints, calculated as the mean changes in measurements between baseline and the end of the 16-wk intervention for 1) plasma α-tocopherol, 2) urinary α-carboxyethyl hydroxychromanol (α-CEHC; an α-tocopherol metabolite), and 3) serum magnesium. Results: Hazelnut consumption increased concentrations of the urinary α-tocopherol metabolite α-CEHC (mean ± SD: 0.84 ± 0.45 to 1.14 ± 0.50 µmol/g creatinine; P = 0.0006). In addition, hazelnut consumption increased serum concentrations of magnesium (+2.1%, P = 0.05), decreased concentrations of fasting glucose (-3.4%, P = 0.03) and LDL cholesterol (-6.0%, P = 0.02), and decreased total:HDL cholesterol ratios (-4.5%, P = 0.009). No significant changes were observed in blood pressure, lymphocyte proliferation assays, and serum concentrations of insulin, high-sensitivity C-reactive protein, triglyceride, α-tocopherol, or HDL cholesterol. Conclusions: Consuming hazelnuts improves a biomarker of vitamin E status in older adults. Vitamin E is a shortfall micronutrient, as identified by the Dietary Guidelines for Americans 2015-2020, which frequently is consumed at levels less than the Estimated Average Requirement of 12 mg/d; thus, hazelnuts should be considered as part of a healthy dietary pattern. This trial was registered at clinicaltrials.gov as NCT03485989.

Metabolomic analysis reveals distinct profiles in the plasma and urine of rats fed a high-protein diet.

A high-protein, low-carbohydrate diet has been regarded as a dietary intervention for weight loss in the obese population. We integrated metabolomics profiles and correlation-based network analysis to reveal the difference in metabolism under diets with different protein:carbohydrate ratios. Rats were fed a control diet (moderate-protein moderate-carbohydrate: MPMC; 20 % protein, 56 % carbohydrate) or HPLC diet (high-protein low-carbohydrate: 45 % protein, 30 % carbohydrate) for 6 weeks. The fat content was equal for both diets. HPLC feeding induced weight loss and reduced adipose weight and plasma triglyceride. Compared to the MPMC diet, HPLC significantly increased plasma α-tocopherol, pyruvate, 2-oxoisocaproate, and ß-hydroxybutyrate, and reduced linoleate, palmitate, α-glycerophosphate and pyroglutamic acid. The HPLC-associated urinary metabolite profile was signified with an increase in palmitate and stearate and a reduction of citrate, 2-ketoglutarate, malate, and pantothenate. Pathway analysis implicated a significant alteration of the TCA cycle in urine. Biomarker screening demonstrated that individual metabolites, including plasma urea, pyruvate, and urinary citrate, robustly distinguished the HPLC group from the MPMC group. Correlation-based network analysis enabled to demonstrate that the correlation of plasma metabolite was strengthened after the HPLC diet, while the energy-metabolism relatives 2-ketoglutarate and fumarate correlated positively with phenylalanine, methionine, and serine. The correlation network between plasma-urinary metabolites revealed a negative correlation of plasma valine with urinary ß-hydroxybutyrate in MPMC rats. In HPLC rats, plasma 2-oxoisocaproate negatively correlated with urinary pyruvate and glycine. This study using metabolomics analysis revealed the systemic metabolism in response to diet treatment and identified the significantly distinct profiles associated with a HPLC diet.

α-Tocopherol does not accelerate depletion of γ-tocopherol and tocotrienol or excretion of their metabolites in rats.

From an enzyme kinetic study using rat liver microsomes, α-tocopherol has been suggested to accelerate the other vitamin E catabolism by stimulating vitamin E ω-hydroxylation, the late limiting reaction of the vitamin E catabolic pathway. To test the effect of α-tocopherol on catabolism of the other vitamin E isoforms in vivo, we determined whether α-tocopherol accelerates depletion of γ-tocopherol and tocotrienol and excretion of their metabolites in rats. Male Wistar rats were fed a γ-tocopherol-rich diet for 6 weeks followed by a γ-tocopherol-free diet with or without α-tocopherol for 7 days. Intake of γ-tocopherol-free diets lowered γ-tocopherol concentrations in serum, liver, adrenal gland, small intestine, and heart, but there was no effect of dietary α-tocopherol on γ-tocopherol concentrations. The level of urinary excretion of γ-tocopherol metabolite was not affected by dietary α-tocopherol. Next, the effect of α-tocopherol on tocotrienol depletion was examined using rats fed a tocotrienol-rich diet for 6 weeks. Subsequent intake of a tocotrienol-free diet with or without α-tocopherol for 7 days depleted concentrations of α- and γ-tocotrienol in serum and tissues, which was accompanied by a decrease in the excretion of γ-tocotrienol metabolite. However, neither the tocotrienol concentration nor γ-tocotrienol metabolite excretion was affected by dietary α-tocopherol. These data showed that dietary α-tocopherol did not accelerate the depletion of γ-tocopherol and tocotrienol and their metabolite excretions, suggesting that the positive effect of α-tocopherol on vitamin E ω-hydroxylase is not sufficient to affect the other isoform concentrations in tissues.

Cytochrome P450 regulation by α-tocopherol in Pxr-null and PXR-humanized mice.

The pregnane X receptor (PXR) has been postulated to play a role in the metabolism of α-tocopherol owing to the up-regulation of hepatic cytochrome P450 (P450) 3A in human cell lines and murine models after α-tocopherol treatment. However, in vivo studies confirming the role of PXR in α-tocopherol metabolism in humans presents significant difficulties and has not been performed. PXR-humanized (hPXR), wild-type, and Pxr-null mouse models were used to determine whether α-tocopherol metabolism is influenced by species-specific differences in PXR function in vivo. No significant difference in the concentration of the major α-tocopherol metabolites was observed among the hPXR, wild-type, and Pxr-null mice through mass spectrometry-based metabolomics. Gene expression analysis revealed significantly increased expression of Cyp3a11 as well as several other P450s only in wild-type mice, suggesting species-specificity for α-tocopherol activation of PXR. Luciferase reporter assay confirmed activation of mouse PXR by α-tocopherol. Analysis of the Cyp2c family of genes revealed increased expression of Cyp2c29, Cyp2c37, and Cyp2c55 in wild-type, hPXR, and Pxr-null mice, which suggests PXR-independent induction of Cyp2c gene expression. This study revealed that α-tocopherol is a partial agonist of PXR and that PXR is necessary for Cyp3a induction by α-tocopherol. The implications of a novel role for α-tocopherol in Cyp2c gene regulation are also discussed.

Vitamin E decreases extra-hepatic menaquinone-4 concentrations in rats fed menadione or phylloquinone.

SCOPE: The mechanism for increased bleeding and decreased vitamin K status accompanying vitamin E supplementation is unknown. We hypothesized that elevated hepatic α-tocopherol (α-T) concentrations may stimulate vitamin K metabolism and excretion. Furthermore, α-T may interfere with the side chain removal of phylloquinone (PK) to form menadione (MN) as an intermediate for synthesis of tissue-specific menaquinone-4 (MK-4). METHODS AND RESULTS: In order to investigate these hypotheses, rats were fed phylloquinone (PK) or menadione (MN) containing diets (2 µmol/kg) for 2.5 weeks. From day 10, rats were given daily subcutaneous injections of either α-T (100 mg/kg) or vehicle and were sacrificed 24 h after the seventh injection. Irrespective of diet, α-T injections decreased MK-4 concentrations in brain, lung, kidney, and heart; and PK in lung. These decreases were not accompanied by increased excretion of urinary 5C- or 7C-aglycone vitamin K metabolites, however, the urinary α-T metabolite (α-CEHC) increased ≥ 100-fold. Moreover, α-T increases were accompanied by downregulation of hepatic cytochrome P450 expression and modified expression of tissue ATP-binding cassette transporters. CONCLUSION: Thus, in rats, high tissue α-T depleted tissue MK-4 without significantly increasing urinary vitamin K metabolite excretion. Changes in tissue MK-4 and PK levels may be a result of altered regulation of transporters.

Quantitation of [5-14CH3]-(2R, 4'R, 8'R)-α-tocopherol in humans.

Half-lives of α-tocopherol in plasma have been reported as 2-3 d, whereas the Elgin Study required >2 y to deplete α-tocopherol, so gaps exist in our quantitative understanding of human α-tocopherol metabolism. Therefore, 6 men and 6 women aged 27 ± 6 y (mean ± SD) ingested 1.81 nmol, 3.70 kBq of [5-(14)CH(3)]-(2R, 4'R, 8'R)-α-tocopherol. The levels of (14)C in blood plasma and washed RBC were monitored frequently from 0 to 460 d while the levels of (14)C in urine and feces were monitored from 0 to 21 d. Total fecal elimination (fecal + metabolic fecal) was 23.24 ± 5.81% of the (14)C dose, so feces over urine was the major route of elimination of the ingested [5-(14)CH(3)]-(2R, 4'R, 8'R)-α-tocopherol, consistent with prior estimates. The half-life of α-tocopherol varied in plasma and RBC according to the duration of study. The minute dose coupled with frequent monitoring over 460 d and 21 d for blood, urine, and feces ensured the [5-(14)CH(3)]-(2R, 4'R, 8'R)-α-tocopherol (the tracer) had the chance to fully mix with the endogenous [5-(14)CH(3)]-(2R, 4'R, 8'R)-α-tocopherol (the tracee). The (14)C levels in neither plasma nor RBC had returned to baseline by d 460, indicating that the t(1/2) of [5-CH(3)]-(2R, 4'R, 8'R)-α-tocopherol in human blood was longer than prior estimates.

Rapid HPLC method for the determination of vitamin A and E and cotinine concentration in human serum in women with CIN and cervical cancer.

OBJECTIVE: The aim of this study was to elaborate on the analytical method for quantitative determination of retinol and alpha-tocopherol in serum of women diagnosed with CIN and cervical cancer. The basic problem in the analysis of the vitamins content in biological material is their low physiological concentration level and instability. Liquid chromatography with diode array detector (DAD) was applied. MATERIAL AND METHODS: The material consisted of serum and urine collected from 12 women diagnosed with cervical intraepithelial neoplasia (CIN) and 16 diagnosed with cervical cancer. The method was evaluated for the following parameters: linearity, recovery, sensitivity, precision, accuracy, selectivity, stability, limit of quantification (LOQ) and limit of detection (LOD). RESULTS: Results showed good linearity (r2> or =0.99) in the range 0.1 microg/ml-10 mg/ml for retinol and 0.25 microg/ml-15 microg/ml for alpha-tocopherol. The Lower Limit of Detection was 0.15 microg/ml for vitamin E and 0.05 microg/ml for vitamin A. The within-run R.S.Ds were below 5.2% at all concentration levels and the between-run R.S.Ds were below 10.0% at all concentration levels. CONCLUSIONS: The advantage of this method is that it measures both compounds in a more rapid, reproducible and accurate manner when compared to the previous HPLC studies. The compounds (vitamin A and E and internal standards) are measured in the same sample at the same time. Quantitative determination of cotinine may reveal active smokers and subjects exposed to environmental tobacco smoke, which is independent measurable carcinogenetic co-factor. The following study is a part of a project determining non-viral causative agents in cervical carcinogenesis.

[Analysis of primary metabolites of alpha-tocopherol in human urine by liquid chromatography-mass spectrometry].

To investigate primary metabolites of alpha-tocopherol in human urine, the urine samples of five healthy volunteers after oral administration of 250 mg vitamin E once a day for seven days were collected within 0 -6 h in the seventh day. The samples were purified through C18 solid-phase extraction cartridge and analyzed by liquid chromatography-tandem mass spectrometry. alpha-Tocopheronic acid, 2,5,7, 8-tetramethyl-2-(2'-carboxyethyl) -6-suphate-chroman (alpha-CEHC-sulphate), gamma-tocopheronolactone, and 2, 5, 7, 8-tetramethyl-2-(4', 8', 12'-trimethyl-12'-carboxy dodecanyl) -6-suphate-chroman were found in urine of volunteers as four primary metabolites of alpha-tocopherol. The method has shown to be promising for alpha-tocopherol detection with many desirable properties including high sensitivity and selectivity, thus providing a reliable pathway for further study in metabolism of alpha-tocopherol.

A feasibility study quantifying in vivo human alpha-tocopherol metabolism.

BACKGROUND: Quantitation of human vitamin E metabolism is incomplete, so we quantified RRR- and all-rac-alpha-tocopherol metabolism in an adult. OBJECTIVE: The objective of the study was to quantify and interpret in vivo human vitamin E metabolism. DESIGN: A man was given an oral dose of 0.001821 micromol [5-14CH3]RRR-alpha-tocopheryl acetate (with 101.5 nCi 14C), and its fate in plasma, plasma lipoproteins, urine, and feces was measured over time. Data were analyzed and interpreted by using kinetic modeling. The protocol was repeated later with 0.001667 micromol [5-14CH3]all-rac-alpha-tocopheryl acetate (with 99.98 nCi 14C). RESULTS: RRR-alpha-tocopheryl acetate and all-rac-alpha-tocopheryl acetate were absorbed equally well (fractional absorption: approximately 0.775). The main route of elimination was urine, and approximately 90% of the absorbed dose was alpha-2(2'-carboxyethyl)-6-hydroxychroman. Whereas 93.8% of RRR-alpha-tocopherol flow to liver kinetic pool B from plasma was returned to plasma, only 80% of the flow of all-rac-alpha-tocopherol returned to plasma; the difference (14%) was degraded and eliminated. Thus, for newly digested alpha-tocopherol, the all-rac form is preferentially degraded and eliminated over the RRR form. Respective residence times in liver kinetic pool A and plasma for RRR-alpha-tocopherol were 1.16 and 2.19 times as long as those for all-rac-alpha-tocopherol. Model-estimated distributions of plasma alpha-tocopherol, extrahepatic tissue alpha-tocopherol, and liver kinetic pool B for RRR-alpha-tocopherol were, respectively, 6.77, 2.71, and 3.91 times as great as those for all-rac-alpha-tocopherol. Of the lipoproteins, HDL had the lowest 14C enrichment. Liver had 2 kinetically distinct alpha-tocopherol pools. CONCLUSIONS: Both isomers were well absorbed; all-rac-alpha-tocopherol was preferentially degraded and eliminated in urine, the major route. RRR-alpha-tocopherol had a longer residence time and larger distribution than did all-rac-alpha-tocopherol. Liver had 2 distinct alpha-tocopherol pools. The model is a hypothesis, its estimates are model-dependent, and it encourages further testing.

Assessment of tocopherol metabolism and oxidative stress in familial hypobetalipoproteinemia.

BACKGROUND: Vitamin E supplementation has been recommended for persons with familial hypobetalipoproteinemia (FHBL), a rare disorder of lipoprotein metabolism that leads to low serum alpha-tocopherol and decreased LDL-cholesterol and apolipoprotein (apo) B. We examined the effect of truncated apoB variants on vitamin E metabolism and oxidative stress in persons with FHBL. METHODS: We studied 9 individuals with heterozygous FHBL [mean (SE) age, 40 (5) years; body mass index (BMI), 27 (10) kg/m2] and 7 normolipidemic controls [age, 41 (5) years; BMI, 25 (2) kg/m2]. We also studied 3 children-2 with homozygous FHBL (apoB-30.9) and 1 with abetalipoproteinemia-who were receiving alpha-tocopherol supplementation. We used HPLC with electrochemical detection to measure alpha- and gamma-tocopherol in serum, erythrocytes, and platelets, and gas chromatography-mass spectrometry to measure F2-isoprostanes and tocopherol metabolites in urine as markers of oxidative stress and tocopherol intake, respectively. RESULTS: Compared with controls, persons with FHBL had significantly lower fasting plasma concentrations of total cholesterol [2.4 (0.2) vs 4.7 (0.2) mmol/L], triglycerides [0.5 (0.1) vs 0.9 (0.1) mmol/L], LDL-cholesterol [0.7 (0.1) vs 2.8 (0.3) mmol/L], apoB [0.23 (0.02) vs 0.84 (0.08) g/L], alpha-tocopherol [13.6 (1.0) vs 28.7 (1.4) micromol/L], and gamma-tocopherol [1.0 (0.1) vs 1.8 (0.3) micromol/L] (all P < 0.03). Erythrocyte alpha-tocopherol was decreased [5.0 (0.2) vs 6.0 (0.3) micromol/L; P < 0.005], but we observed no differences in lipid-adjusted serum tocopherols, erythrocyte gamma-tocopherol, platelet alpha- or gamma-tocopherol, urinary F2-isoprostanes, or tocopherol metabolites. CONCLUSION: Taken together, our findings do not support the recommendation that persons with heterozygous FHBL receive vitamin E supplementation.

The effect of gamma-tocopherol administration on alpha-tocopherol levels and metabolism in humans.

BACKGROUND: The bioavailability of gamma-tocopherol and metabolites of vitamin E after gamma-tocopherol administration is not well understood. We investigated the effect of gamma-tocopherol administration on the levels and metabolism of alpha- and gamma-tocopherol in healthy volunteers. METHODS: We measured two metabolites of vitamin E (2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC) and 2,7,8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman (gamma-CEHC)) in plasma and urine by high-performance liquid chromatography with electrochemical detection (HPLC-ECD) during administration of gamma-tocopherol. Two groups of volunteers were enrolled. The gamma-tocopherol group received two gamma-tocopherol capsules (each containing 186.4 mg of gamma-tocopherol and 5 mg of alpha-tocopherol) for 28 days, while the control group received d-alpha-tocopherol at 5 mg/day, which was the same dose as that given to the gamma-tocopherol group. Blood and urine samples were obtained on days 0, 14, 28, 35, 42, and 56 after the initiation of gamma-tocopherol administration. RESULTS: The plasma gamma-tocopherol concentration increased markedly during administration of gamma-tocopherol and the plasma gamma-CEHC concentration increased along with that of gamma-tocopherol. The plasma alpha-tocopherol concentration decreased significantly during gamma-tocopherol administration. The plasma concentration of alpha-CEHC decreased significantly and urinary excretion of alpha-CEHC tended to increase in the gamma-tocopherol group. Urinary sodium secretion was significantly increased at 1 week after the cessation of gamma-tocopherol administration, but there was no significant difference of urine volume between the two groups. CONCLUSION: Metabolism of alpha-tocopherol is accelerated and the plasma alpha-tocopherol concentration is decreased during gamma-tocopherol administration.

{alpha}-Tocopherol disappearance is faster in cigarette smokers and is inversely related to their ascorbic acid status.

BACKGROUND: Cigarette smokers have enhanced oxidative stress from cigarette smoke exposure and from their increased inflammatory responses. OBJECTIVE: The objective of this study was to determine whether cigarette smoking increases plasma alpha-tocopherol disappearance in otherwise healthy humans. DESIGN: Smokers and nonsmokers (n = 10/group) were supplemented with deuterium-labeled alpha-tocopheryl acetates (75 mg each of d(3)-RRR-alpha-tocopheryl acetate and d(6)-all-rac-alpha-tocopherols acetate) for 6 evenings (days -6 to -1). Plasma alpha-tocopherols, ascorbic acid, uric acid, and F(2alpha)-isoprostanes were measured in blood samples collected on days -6 through 17. The urinary alpha-tocopherol metabolite, alpha-carboxy-ethyl-hydroxy-chroman (alpha-CEHC), was measured on days -6, 0, and 17 in 24-h urine samples. RESULTS: F(2alpha)-isoprostanes were, on average, approximately 40% higher in smokers than in nonsmokers. On day 0, plasma labeled and unlabeled alpha-tocopherol concentrations were not significantly different between groups. Smoking resulted in faster fractional disappearance of plasma alpha-tocopherol (0.215 +/- 0.011 compared with 0.191 +/- 0.009 pools/d; P < 0.05). Fractional disappearance rates of alpha-tocopherol correlated with plasma ascorbic acid concentrations in smokers (P = 0.021) but not in nonsmokers despite plasma ascorbic acid concentrations that were not significantly different between groups. By day 17, cigarette smoking resulted in lower plasma alpha-tocopherol concentrations and urinary excretion of labeled and unlabeled alpha-CEHC (P < 0.05). CONCLUSIONS: Cigarette smoking increased alpha-tocopherol disappearance. Greater rates of alpha-tocopherol disappearance in smokers appear to be related to increased oxidative stress accompanied by lower plasma ascorbic acid concentrations. Thus, smokers have an increased requirement for both alpha-tocopherol and ascorbic acid.

Oxidative stress in a rat model of nephrosis can be quantified by electron spin resonance.

The pathogenesis of nephrotic syndrome is not clear. In this study, we used electron spin resonance (ESR) to evaluate levels of reactive oxygen species in rats with puromycin aminonucleoside (PAN)-induced nephrosis. Twenty-six Wistar rats were divided into four groups: (1) PAN treated, (2) PAN treated and alpha-tocopherol supplemented, (3) supplemented with alpha-tocopherol only, (4) control. On day 9, urinary protein excretion was measured. On day 10, all animals were sacrificed with retrograde perfusion via the aorta to obtain renal venous perfusates. The signal intensities of ascorbate radicals in the perfusates were determined by ESR. After perfusion, the kidneys were isolated and sieved to obtain glomeruli for determination of glomerular thiobarbituric acid-reactive substance (TBArs) and alpha-tocopherol. Urinary protein excretion by PAN-treated rats increased significantly on day 9 and was reduced by alpha-tocopherol supplementation. The ascorbate radical intensity and glomerular TBArs level were higher in PAN-treated than in control rats and were both suppressed to control levels by alpha-tocopherol supplementation. There were positive correlations between ascorbate radical intensity and the daily urinary protein, as well as between ascorbate radical intensity and the glomerular TBArs level. Hence, it is possible to quantify oxidative stress due to PAN nephrosis by ESR. Our findings suggest that lipid peroxidation plays an important role in the pathogenesis of proteinuria in PAN-treated rats.

Inter- and intra-individual vitamin E uptake in healthy subjects is highly repeatable across a wide supplementation dose range.

Vitamin E uptake after supplementation varies widely in the healthy population, and preliminary studies have indicated that individual responses are relatively stable over periods in excess of 1 year. This phenotypic stability suggests a genetic basis to this observed variation. To examine this issue further, we examined the repeatability of both baseline plasma alpha-tocopherol and urinary alpha-tocopherol metabolite concentrations, as well as individual responses of these parameters after vitamin E supplementation. In the first study, 65 subjects (33 males, 32 females, aged 30.7 +/- 7.4 years) provided three plasma and urine samples for alpha-tocopherol and metabolite analysis with each collection separated by at least 2 weeks. Plasma alpha-tocopherol concentrations were found to be highly repeatable over this short interval (intra-class correlation coefficient [ICC] = 0.85), although the association deteriorated once values were corrected for plasma cholesterol (ICC = 0.64). Similarly, urinary alpha-tocopherol metabolites 2(2'-carboxyethyl)-6-hydroxychroman acid (alpha-CEHC) and quinone lactone (QL) concentration were found to display a moderate degree of intra-subject repeatability: ICC = 0.65 and 0.58, respectively. In a second study, plasma alpha-tocopherol and urinary metabolite responses were investigated in 18 healthy, nonsmoking subjects (12 males, 6 females, aged 33.1 +/- 9.1 years) after successive 6-week periods of vitamin E (RRR-alpha-tocopherol acetate) supplementation at 15, 100, 200, and 400 mg/day. Plasma and urine samples were obtained on days 0, 7, 14, 21, and 28 (7 days after the final supplement) of each dosing period and the strength of the underlying association between responses determined using Kendall's tau_b test. Individual plasma alpha-tocopherol responses at the 100, 200, and 400 mg/day doses were found to be highly associated: tau, 0.51, P = 0.02 [100 vs. 200] and tau, 0.49, P = 0.03 [100 vs. 400] and tau, 0.56, P = 0.005 [200 vs. 400]. Together these data support the contention that alpha-tocopherol uptake is a stable individual phenotype under genetic regulation.

The effect of age on vitamin E status, metabolism, and function: metabolism as assessed by labeled tocopherols.

The effects of age on vitamin E metabolism were studied in 97 healthy 20-75-year-old male nonsmoking Austrian volunteers of the VITAGE project. After a single oral intake of 30 mg d(6)-RRR-alpha- and d(2)-RRR-gamma-tocopheryl acetate, blood and 24-hour urine was collected. Deuterated tocopherols in plasma and deuterated urinary metabolites were analyzed by GC-MS. A first evaluation revealed a similar uptake of d(6)-alpha- and d(2)-gamma-tocopherol during the first 6 hours, and then d(2)-gamma-tocopherol started to decrease. Urinary d(2)-gamma- carboxyethyl hydroxychroman metabolites (CEHCs) exceeded those of d(6)-alpha-CEHCs by about 10 times. There was no effect of age. Thus, there might be no need for a higher vitamin E intake for healthy elderly nonsmoking men.

gamma-Tocopherol biokinetics and transformation in humans.

BACKGROUND: The uptake and biotransformation of gamma-tocopherol (gamma-T) in humans is largely unknown. Using a stable isotope method we investigated these aspects of gamma-T biology in healthy volunteers and their response to gamma-T supplementation. METHODS: A single bolus of 100 mg of deuterium labeled gamma-T acetate (d(2)-gamma-TAC, 94% isotopic purity) was administered with a standard meal to 21 healthy subjects. Blood and urine (first morning void) were collected at baseline and a range of time points between 6 and 240 h post-supplemetation. The concentrations of d(2) and d(0)-gamma-T in plasma and its major metabolite 2,7,8-trimethyl-2-(b-carboxyethyl)-6-hydroxychroman (-gamma-CEHC) in plasma and urine were measured by GC-MS. In two subjects, the total urine volume was collected for 72 h post-supplementation. The effects of gamma-T supplementation on alpha-T concentrations in plasma and alpha-T and gamma-T metabolite formation were also assessed by HPLC or GC-MS analysis. RESULTS: At baseline, mean plasma alpha-T concentration was approximately 15 times higher than gamma-T (28.3 vs. 1.9 micromol/l). In contrast, plasma gamma-CEHC concentration (0.191 micromol/l) was 12 fold greater than alpha-CEHC (0.016 micromol/l) while in urine it was 3.5 fold lower (0.82 and 2.87 micromol, respectively) suggesting that the clearance of alpha-CEHC from plasma was more than 40 times that of gamma-CEHC. After d(2)-gamma-TAC administration, the d(2) forms of gamma-T and gamma-CEHC in plasma and urine increased, but with marked inter-individual variability, while the d(0) species were hardly affected. Mean total concentrations of gamma-T and gamma-CEHC in plasma and urine peaked, respectively, between 0-9, 6-12 and 9-24 h post-supplementation with increases over baseline levels of 6-14 fold. All these parameters returned to baseline by 72 h. Following challenge, the total urinary excretion of d(2)-gamma-T equivalents was approximately 7 mg. Baseline levels of gamma-T correlated positively with the post-supplementation rise of (d(0) + d(2)) - gamma - T and gamma-CEHC levels in plasma, but correlated negatively with urinary levels of (d(0) + d(2))-gamma-CEHC. Supplementation with 100 mg gamma-TAC had minimal influence on plasma concentrations of alpha-T and alpha-T-related metabolite formation and excretion. CONCLUSIONS: Ingestion of 100mg of gamma-TAC transiently increases plasma concentrations of gamma-T as it undergoes sustained catabolism to CEHC without markedly influencing the pre-existing plasma pool of gamma-T nor the concentration and metabolism of alpha-T. These pathways appear tightly regulated, most probably to keep high steady-state blood ratios alpha-T to gamma-T and gamma-CEHC to alpha-CEHC.