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Background: Urinary metabolites of vitamin E, i.e., α- and γ-carboxyethyl hydroxychroman (α- and γ-CEHC), have gained increasing attention and have been proposed as novel biomarkers of vitamin E intake and status. However, there are insufficient data on the relationship of plasma α-tocopherol and γ-tocopherol and dietary vitamin E intake with 24 h urinary excretions of α- and γ-CEHC., Objectives: We aimed to (1) investigate the associations of urinary α- and γ-CEHC/creatinine ratios and 24 h urinary excretions of α- and γ-CEHC with plasma α- and γ-tocopherol, respectively; (2) investigate the associations of urinary α- and γ-CEHC/creatinine ratios and 24 h urinary excretions of α- and γ-CEHC with dietary vitamin E intake, and we hypothesize that 24 h urinary excretions of α- and γ-CEHC will better correlate with vitamin E intake than urinary α- and γ-CEHC/creatinine ratios., Design: 24 h Urine and plasma samples were collected from 1519 participants (60-75 years, male: 50%) included in the Lifelines-MINUTHE Study for the assessments of urinary α- and γ-CEHC/creatinine ratios and 24 h urinary excretions of α- and γ-CEHC, and plasma α- and γ-tocopherol. Among those participants, dietary vitamin E intake data from 387 participants were available from an externally validated Flower-Food Frequency Questionnaire (FFQ). The associations of plasma α- and γ-tocopherol, dietary vitamin E intake, with urinary α- and γ-CEHC were assessed using multivariate linear regressions., Results: 24 h Urinary excretion of α-CEHC (median (IQR): 0.9 (0.3-2.4) µmol) was less than that of γ-CEHC (median (IQR): 1.5 (0.5-3.5) µmol). After adjustment for covariates, we found that 24 h urinary α-CEHC excretion and urinary α-CEHC/creatinine ratio were both positively associated with plasma α-tocopherol (std.beta: 0.06, p = 0.02; std.beta: 0.06, p = 0.01, respectively). Furthermore, the sum of 24 h urinary α- and γ-CEHC excretions was positively associated with dietary vitamin E intake (std.beta: 0.08; p = 0.03), whereas there was no relation between urinary α- and γ-CEHC/creatinine ratios and vitamin E intake. No association was observed neither between plasma α- and γ-tocopherol and dietary vitamin E intake, nor between urinary γ-CEHC and plasma γ-tocopherol., Conclusion: Our study confirmed our hypothesis that 24 h urinary α- and γ-CEHC excretions would be a better marker for dietary vitamin E intake than urinary α- and γ-CEHC/creatinine ratios. Considering that both 24 h urinary α- and γ-CEHC excretions and α- and γ-CEHC/creatinine ratios were also associated with plasma α-tocopherol status, we suggest that 24 h urinary α- and γ-CEHC excretions could be used to assess overall vitamin E status., (© 2022. The Author(s).)

Background & Aims: Damage induced by lipid peroxidation has been associated with impaired glucose homeostasis. Vitamin E (α-tocopherol, α-TOH) competitively reacts with lipid peroxyl radicals to mitigate oxidative damage, and forms oxidized vitamin E metabolites. Accordingly, we aimed to investigate the associations between α-TOH metabolites (oxidized and enzymatic) in both circulation and urine and measures of glucose homeostasis in the general middle-aged population., Methods: This cross-sectional study was embedded in the population-based Netherlands Epidemiology of Obesity (NEO) Study. α-TOH metabolites in blood (α-TOH and α-CEHC-SO 3 ) and urine [sulfate (SO 3 ) and glucuronide (GLU) of both α-TLHQ (oxidized) and α-CEHC (enzymatic)] were quantified by liquid chromatography coupled with tandem mass spectrometry (LC/MS-MS). Measures of glucose homeostasis (HOMA-B, HOMA-IR, Insulinogenic index and Matsuda index) were obtained from fasting and postprandial blood samples. Multivariable linear regression analyses were performed to assess the associations of α-TOH metabolites and measures of glucose homeostasis., Results: We included 498 participants (45% men) with mean (SD) age of 55.8 (6.1) years who did not use glucose-lowering medication. While blood α-TOH was not associated with measures of glucose homeostasis, urinary oxidized metabolites (α-TLHQ-SO 3 /GLU) were associated with HOMA-IR and Matsuda index. For example, a one-SD higher α-TLHQ-SO 3 was associated with 0.92 (95% CI: 0.87, 0.97) fold lower HOMA-IR and 1.06 (1.01, 1.11) fold higher Matsuda index, respectively. Similar results were obtained for the urinary α-TLHQ to α-CEHC ratio as a measure of oxidized-over-enzymatic conversion of α-TOH., Conclusion: Higher urinary levels of oxidized α-TOH metabolites as well as higher oxidized-to-enzymatic α-TOH metabolite ratio, but not circulating α-TOH or enzymatic metabolites, were associated with lower insulin resistance. Rather than circulating α-TOH, estimates of the conversion of α-TOH might be informative in relation to health and disease., Competing Interests: Conflicts of interest Dr. Mook-Kanamori is a part time research consultant for Metabolon, Inc. The other authors declare no conflict of interest., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: α-Tocopherol (αT) in its natural form [2'R, 4'R, 8'R αT (RRR-αT)] is more bioactive than synthetic α-tocopherol (all rac-αT). All rac-αT is widely used in infant formulas, but its accretion in formula-fed infant brain is unknown., Objective: We sought to compare αT and stereoisomer status in infant rhesus macaques (Macaca mulatta) fed infant formula (RRR-αT or all rac-αT) with a reference group fed a mixed diet of breast milk and maternal diet., Methods: From 1 d after birth until 6 mo of age, infants (n = 23) were either nursery reared and exclusively fed 1 of 2 formulas by staff personnel or were community housed with their mothers and consumed a mixed reference diet of breast milk (69 mL/d at 6 mo) transitioning to monkey diet at ∼2 mo (MF; n = 8). Formulas contained either 21 μmol RRR-αT/L (NAT-F; n = 8) or 30 μmol all rac-αT/L (SYN-F; n = 7). Total αT and αT stereoisomers were analyzed in breast milk at 2, 4, and 6 mo and in monkey plasma and liver and 6 brain regions at 6 mo of age. α-Tocopherol transfer protein (α-TTP), lipoprotein αT, and urinary α-carboxyethyl-hydroxychroman (α-CEHC) were measured. One-way ANOVA with Tukey's post-hoc test was used for analysis., Results: At study termination, plasma, liver, lipoprotein, and brain total αT did not differ between groups. However, the NAT-F-fed group had higher RRR-αT than the SYN-F-fed group (P < 0.01) and the MF group (P < 0.0001) in plasma (1.7- and 2.7-fold) and brain (1.5- and 2.5-fold). Synthetic αT 2R stereoisomers (SYNTH-2R) were generally 3- and 7-fold lower in brain regions of the NAT-F group compared with those of the SYN-F and MF groups (P < 0.05). SYNTH-2R stereoisomers were 2-fold higher in MF than SYN-F (P < 0.0001). The plasma percentage of SYNTH-2R was negatively correlated with the brain percentage of RRR-αT (r = -0.99, P < 0.0001). Brain αT profiles were not explained by α-TTP mRNA or protein expression. Urine α-CEHC was 3 times higher in the NAT-F than in the MF group (P < 0.01)., Conclusions: Consumption of infant formulas with natural (NAT-F) compared with synthetic (SYN-F) αT differentially impacted brain αT stereoisomer profiles in infant rhesus macaques. Future studies should assess the functional implications of αT stereoisomer profiles on brain health., (Copyright © The Author(s) on behalf of the American Society for Nutrition 2020.)

In rats, plasma and tissue concentrations of α-tocopherol, a predominant form of vitamin E in mammals, are known to differ between the sexes. In order to examine sex differences in α-tocopherol metabolism, we investigated urinary excretion of the α-tocopherol metabolite α-carboxymethylhydroxychroman (α-CEHC) using Wistar rats. First, we measured α-CEHC in urine of 9-week-old male and female rats in basal and α-tocopherol-administered conditions. We observed that female rats excrete significantly more α-CEHC than male rats via urine. This sex difference was observed in matured 9-week-old rats but not in premature 3-week-old rats, suggesting that the difference may relate to sex hormones. In order to confirm this, we examined the effect of ovariectomy and orchiectomy on female and male rats, respectively. The results of castration clearly demonstrated that orchiectomy enhanced urinary excretion of α-CEHC, supporting the hypothesis that testosterone repressed α-tocopherol metabolism. We then administered testosterone propionate to orchiectomized rats and observed down-regulation of α-CEHC excretion. Taken together, these results indicate that testosterone represses the metabolism and urinary excretion of α-tocopherol in rats. This is the first report to show a sex-dependent difference in urinary excretion rate of an α-tocopherol metabolite and contributes to the understanding of vitamin E metabolism., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Vitamin E supplementation improves liver histology in patients with nonalcoholic steatohepatitis, which is a manifestation of the metabolic syndrome (MetS). We reported previously that α-tocopherol bioavailability in healthy adults is higher than in those with MetS, thereby suggesting that the latter group has increased requirements. Objective: We hypothesized that α-tocopherol catabolites α-carboxyethyl hydroxychromanol (α-CEHC) and α-carboxymethylbutyl hydroxychromanol (α-CMBHC) are useful biomarkers of α-tocopherol status. Design: Adults (healthy or with MetS; n = 10/group) completed a double-blind, crossover clinical trial with four 72-h interventions during which they co-ingested 15 mg hexadeuterium-labeled RRR -α-tocopherol (d 6 -α-T) with nonfat, reduced-fat, whole, or soy milk. During each intervention, we measured α-CEHC and α-CMBHC excretions in three 8-h urine collections (0-24 h) and plasma α-tocopherol, α-CEHC, and α-CMBHC concentrations at various times ≤72 h. Results: During the first 24 h, participants with MetS compared with healthy adults excreted 41% less α-CEHC (all values are least-squares means ± SEMs: 0.6 ± 0.1 compared with 1.0 ± 0.1 μmol/g creatinine, respectively; P = 0.002), 63% less hexadeuterium-labeled (d 6 )-α-CEHC (0.04 ± 0.02 compared with 0.13 ± 0.02 μmol/g creatinine, respectively; P = 0.002), and 58% less d 6 -α-CMBHC (0.017 ± 0.004 compared with 0.041 ± 0.004 μmol/g creatinine, respectively; P = 0.0009) and had 52% lower plasma d 6 -α-CEHC areas under the concentration curves [area under the curve from 0 to 24 h (AUC 0-24h ): 27.7 ± 7.9 compared with 58.4 ± 7.9 nmol/L × h, respectively; P = 0.01]. d 6 -α-CEHC peaked before d 6 -α-T in 77 of 80 paired plasma concentration curves. Urinary d 6 -α-CEHC 24-h concentrations were associated with the plasma AUC 0-24 h of d 6 -α-T ( r = 0.53, P = 0.02) and d 6 -α-CEHC ( r = 0.72, P = 0.0003), and with urinary d 6 -α-CMBHC ( r = 0.88, P < 0.0001), and inversely with the plasma inflammation biomarkers C-reactive protein ( r = -0.70, P = 0.0006), interleukin-10 ( r = -0.59, P = 0.007), and interleukin-6 ( r = -0.54, P = 0.01). Conclusion: Urinary α-CEHC and α-CMBHC are useful biomarkers to noninvasively assess α-tocopherol adequacy, especially in populations with MetS-associated hepatic dysfunction that likely impairs α-tocopherol trafficking. This trial was registered at clinicaltrials.gov as NCT01787591., (© 2017 American Society for Nutrition.)

A new type of amine-terminated-ionic-liquid-functionalized graphene/multi-walled carbon nanotubes hybrid material (IL-G/MWCNTs) was synthesized and used as an adsorbent in miniaturized pipette tip solid-phase extraction (PT-SPE) coupled with liquid chromatography for the isolation and determination of (Z)-3-(chloromethylene)-6-fluorothiochroman-4-one (CMFT) in urine. Parameters for the preparation of IL-G/MWCNTs and the PT-SPE procedure, including the mass ratio of graphene oxide and oxidized multi-walled carbon nanotubes, the mass ratio of graphene oxide and the ionic liquid, and the type and volume of washing and elution solvents were optimized to achieve higher extraction efficiency. Good linearity of the method was achieved in the range 0.03-5.0μgmL -1 with a coefficient of determination (r 2 ) of 0.9999. The limits of detection and quantification were 0.009 and 0.030μgmL -1 , respectively. The intra- and inter-day precisions, expressed as relative standard deviations (RSDs), were evaluated by performing replicate analyses of samples spiked at 0.1μgmL -1 on the same day (n=6) and over three consecutive days, and were 4.8 and 5.5%, respectively. Recoveries between 73.9 and 93.9% were obtained at three spiking levels, with RSDs≤7.9%. Five batches of the adsorbent were investigated to confirm the reliability of the preparation method., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A new type of ionic liquid-molecularly imprinted polymers (IL-MIPs) synthesized by precipitation polymerization using 1-allyl-3-methylimidazolium bromide and acrylamide as co-functional monomers and (Z)-6-fluoro-3-(hydroxymethylene)-thiochroman-4-one (FHO) as dummy template was successfully applied as a selective adsorbent of pipette tip solid-phase extraction (PT-SPE) for rapid extraction and determination of (Z)-3-(chloromethylene)-6-flourothiochroman-4-one (CFO) in urine. The factors that affected the extraction efficiency including amounts of adsorbent, types of washing solvent, and types and volumes of elution solvent were optimized. Under the optimum conditions of ionic liquid-molecularly imprinted polymers-pipette tip solid-phase extraction (IL-MIPs-PT-SPE) coupled with HPLC-UV, good linearity for CFO was achieved in a range of 0.1-10.0μgmL(-1) (r=0.9999) and the recoveries at three spiked levels were ranged from 94.8% to 103.4% with the relative standard deviations (RSDs) less than 5.7% (n=3)., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Oxidative stress and inflammation persist years after smoking cessation thereby limiting the restoration of vascular endothelial function (VEF). Although short-term smoking cessation improves VEF, no studies have examined co-therapy of antioxidants in combination with smoking cessation to improve VEF. We hypothesized that improvements in γ-tocopherol (γ-T) status during smoking cessation would improve VEF beyond that from smoking cessation alone by decreasing oxidative stress and proinflammatory responses. A randomized, double-blind, placebo-controlled study was conducted in otherwise healthy smokers (22 ± 1 years; mean ± SEM) who quit smoking for 7 days with placebo (n=14) or γ-T-rich supplementation (n=16; 500 mg γ-T/day). Brachial artery flow-mediated dilation (FMD), cotinine, and biomarkers of antioxidant status, oxidative stress, and inflammation were measured before and after 7 days of smoking cessation. Smoking cessation regardless of supplementation similarly decreased plasma cotinine, whereas γ-T-rich supplementation increased plasma γ-T by seven times and its urinary metabolite γ-carboxyethyl hydroxychroman by nine times (P<0.05). Smoking cessation with γ-T-rich supplementation increased FMD responses by 1.3% (P<0.05) beyond smoking cessation alone (4.1 ± 0.6% vs 2.8 ± 0.3%; mean ± SEM). Although plasma malondialdehyde decreased similarly in both groups (P<0.05), plasma oxidized LDL and urinary F2-isoprostanes were unaffected by smoking cessation or γ-T-rich supplementation. Plasma TNF-α and myeloperoxidase decreased (P<0.05) only in those receiving γ-T-rich supplements and these were inversely related to FMD (P<0.05; R=-0.46 and -0.37, respectively). These findings demonstrate that short-term γ-T-rich supplementation in combination with smoking cessation improved VEF beyond that from smoking cessation alone in young smokers, probably by decreasing the proinflammatory mediators TNF-α and myeloperoxidase., (© 2013 Elsevier Inc. All rights reserved.)

Background: Contradictory results from clinical trials that examined the role of vitamin E in chronic disease could be a consequence of interindividual variation, caused by factors such as xenobiotic use. Cometabolism of vitamin E with other pharmaceutical products could affect the bioavailability of the drug. Thus, it is necessary to understand fully the metabolic routes and biological endpoints of vitamin E., Objective: The objective was to uncover novel metabolites and roles of vitamin E in humans and mouse models., Design: Human volunteers (n = 10) were fed almonds for 7 d and then an α-tocopherol dietary supplement for 14 d. Urine and serum samples were collected before and after dosing. C57BL/6 mice (n = 10) were also fed α-tocopherol-deficient and -enriched diets for 14 d. Urine, serum, and feces were collected before and after dosing, and liver samples were collected after euthanization. Ultraperformance liquid chromatography electrospray ionization time-of-flight mass spectrometry and multivariate data analysis tools were used to analyze the samples., Results: Three novel urinary metabolites of α-tocopherol were discovered in humans and mice: α-carboxyethylhydroxychroman (α-CEHC) glycine, α-CEHC glycine glucuronide, and α-CEHC taurine. Another urinary metabolite, α-CEHC glutamine, was discovered in mice after α-CEHC gavage. Increases in liver fatty acids and decreases in serum and liver cholesterol were observed in mice fed the α-tocopherol-enriched diet., Conclusion: Novel metabolites and metabolic pathways of vitamin E were identified by mass spectrometry-based metabolomics and will aid in understanding the disposition and roles of vitamin E in vivo.

Background: Other than the in vitro erythrocyte hemolysis test, no valid biomarkers of vitamin E status currently exist., Objective: We hypothesized that the urinary vitamin E metabolite α-carboxyethyl hydroxychroman (α-CEHC) could serve as a biomarker., Design: The relations between urinary α-CEHC, plasma α-tocopherol, and vitamin E intakes were assessed by using a previously validated multipass, Web-based, 24-h self-administered dietary recall, and we concurrently collected plasma and 24-h urine samples from 233 participants of both sexes., Results: Median vitamin E intakes were 9.7 mg α-tocopherol/d. Intakes were correlated with plasma α-tocopherol (R = 0.40, P < 0.001) and urinary α-CEHC (R = 0.42, P < 0.001); these correlations were essentially unchanged after multivariate adjustments. On the basis of multiple regression analysis, urinary α-CEHC excretion increased by ~0.086 μmol/g creatinine (95% CI: 0.047, 0.125) for every 1-mg (2.3-μmol) increase in dietary α-tocopherol. Urinary α-CEHC excretion remained at a plateau (median: 1.39 μmol/g creatinine) until dietary intakes of α-tocopherol exceeded 9 mg α-tocopherol/d. The inflection point at which vitamin E metabolism increased was estimated to be at an intake of 12.8 mg α-tocopherol/d. Daily excretion of >1.39 μmol α-CEHC/g creatinine is associated with a greater than adequate α-tocopherol status, as evidenced by increased vitamin E metabolism and excretion., Conclusion: Thus, urinary α-CEHC is a valid biomarker of α-tocopherol status that can be used to set a value for the Estimated Adequate Requirement of vitamin E.

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., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pentamethyl-6-chromanol (PMCol), a chromanol-type compound related to vitamin E, was proposed as an anticancer agent with activity against androgen-dependent cancers. In repeat dose-toxicity studies in rats and dogs, PMCol caused hepatotoxicity, nephrotoxicity, and hematological effects. The objectives of this study were to determine the mechanisms of the observed toxicity and identify sensitive early markers of target organ injury by integrating classical toxicology, toxicogenomics, and metabolomic approaches. PMCol was administered orally to male Sprague-Dawley rats at 200 and 2000 mg/kg daily for 7 or 28 days. Changes in clinical chemistry included elevated alanine aminotransferase, total bilirubin, cholesterol and triglycerides-indicative of liver toxicity that was confirmed by microscopic findings (periportal hepatocellular hydropic degeneration and cytomegaly) in treated rats. Metabolomic evaluations of liver revealed time- and dose-dependent changes, including depletion of total glutathione and glutathione conjugates, decreased methionine, and increased S-adenosylhomocysteine, cysteine, and cystine. PMCol treatment also decreased cofactor levels, namely, FAD and increased NAD(P)+. Microarray analysis of liver found that differentially expressed genes were enriched in the glutathione and cytochrome P450 pathways by PMCol treatment. Reverse transcription-polymerase chain reaction of six upregulated genes and one downregulated gene confirmed the microarray results. In conclusion, the use of metabolomics and toxicogenomics demonstrates that chronic exposure to high doses of PMCol induces liver damage and dysfunction, probably due to both direct inhibition of glutathione synthesis and modification of drug metabolism pathways. Depletion of glutathione due to PMCol exposure ultimately results in a maladaptive response, increasing the consumption of hepatic dietary antioxidants and resulting in elevated reactive oxygen species levels associated with hepatocellular damage and deficits in liver function.

The objective is to determine the association between the 24 hour urinary α-tocopherol catabolite, 2,5,7,8-tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman (α-CEHC) and α-tocopherol intake in an intervention and a cross-sectional studies. In the 4-weeks intervention study, Japanese men (n = 10) consumed the test diet in week 1, and the test diet plus varying amounts of α-tocopherol in the three subsequent weeks: 21 μmol/d α-tocopherol in week 2, 63 μmol/d in week 3, and 125 μmol/d in week 4. A significant association between α-tocopherol intake and urinary α-CEHC was observed in this strictly controlled experiment (r = 0.99, p<0.001). In the cross-sectional study, all foods consumed over 4 consecutive days were recorded in 76 free-living young subjects (18-33 years). The association was weak, but a significant relationship was observed (r = 0.29, p<0.05) even in the cross-sectional study. In the cross-sectional study adults, mean estimated α-tocopherol intake calculated by urinary α-CEHC and the excretory ratio was 91% of their mean intake over the 4 days. The results show that urinary α-CEHC level reflected recent α-tocopherol intake in free-living young Japanese adults, and could be used as a measure of intake during the previous few days, both for group means and for individual rankings within a group.

Meta-analyses studies suggest that high-dose vitamin E may be associated with increased mortality in some populations. Vitamin E may increase the production of CYP3A4 in the liver, and this could lead to an increase in drug metabolism, potentially lowering the efficacy of therapeutic drugs. We hypothesized that upregulation of CYP3A4 by alpha-tocopherol (alpha-TOH) would decrease the plasma concentration of the CYP3A4 substrate midazolam. Baseline metabolism of midazolam (1 mg intravenously) was determined in 12 healthy subjects before randomization into 2 groups of 6 to receive either RRR-alpha-TOH (750 IU/d) or placebo for 3 weeks. At completion, subjects were given an additional 1 mg intravenous bolus of midazolam. Plasma midazolam, 1-hydroxy-midazolam, and urinary alpha-TOH metabolite excretion were measured using gas chromatography mass spectrometry. Serum alpha-TOH was measured using high performance liquid chromatography with electrochemical detection. Serum alpha-TOH increased by 100% (P = 0.002) and urinary alpha-TOH metabolite excretion increased 20-fold in the treatment group versus placebo (P = 0.001). There was no effect on the area under time curve of midazolam in subjects taking alpha-TOH compared with placebo. These findings do not support the hypothesis that alpha-TOH supplementation interferes with hepatic CYP3A4-mediated drug metabolism.

Regular consumption of green tea polyphenols (GTP) is thought to reduce the risk of cardiovascular disease (CVD) but has also been associated with liver toxicity. The present trial aimed to assess the safety and potential CVD health beneficial effects of daily GTP consumption. We conducted a placebo-controlled parallel study to evaluate the chronic effects of GTP on liver function and CVD risk biomarkers in healthy men. Volunteers (treatment: n = 17, BMI 26.7 +/- 3.3 kg/m(2), age 41 +/- 9 y; placebo, n = 16, BMI 25.4 +/- 3.3 kg/m(2), age 40 +/- 10 y) consumed for 3 wk 6 capsules per day (2 before each principal meal) containing green tea extracts (equivalent to 714 mg/d GTP) or placebo. At the beginning and end of the intervention period, we collected blood samples from fasting subjects and measured vascular tone using Laser Doppler Iontophoresis. Biomarkers of liver function and CVD risk (including blood pressure, plasma lipids, and asymmetric dimethylarginine) were unaffected by GTP consumption. After treatment, the ratio of total:HDL cholesterol was significantly reduced in participants taking GTP capsules compared with baseline. Endothelial-dependent and -independent vascular reactivity did not significantly differ between treatments. In conclusion, the present data suggests that the daily consumption of high doses of GTP by healthy men for 3 wk is safe but without effects on CVD risk biomarkers other than the total:HDL cholesterol ratio.

2,5,7,8-Tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), the water-soluble metabolite of alpha-tocopherol (alpha-TOH) with a shortened side chain but an intact hydroxychroman structure, has been identified in human urine and are thought to be produced in significant amount at excess intake of alpha-TOH. In previous studies, CEHCs in biological specimens were measured by HPLC, GC-MS or LC-MS, preceded by a hydrolysis procedure using either enzyme or methanolic HCl. In an attempt to analyze alpha-CEHC in rat urine accordingly, we observed that enzyme hydrolysis was relatively inefficient in releasing alpha-CEHC compared to high concentrations of HCl. The HCl releasable alpha-CEHC conjugate was isolated and chemically identified as 6-O-sulfated alpha-CEHC (alpha-CEHC sulfate). Using the synthetic alpha-CEHC sulfate standard, it was found that sulfatase could not hydrolyze to a significant extent. On the other hand, pretreatment with HCl at 60 degrees C in the presence of ascorbate, followed by a one-step ether extraction, not only hydrolyzed the sulfate conjugate completely but also extracted alpha-CEHC with high recovery. The inclusion of ascorbate minimized the conversion of alpha-CEHC to alpha-tocopheronolactone in the HCl pretreatment. A complete procedure for the quantitative analysis of alpha-CEHC including HCl hydrolysis, ether extraction and reverse phase isocratic HPLC-ECD was thus established. In conclusion, alpha-CEHC sulfate was isolated and identified as the HCl-releasable conjugate of alpha-CEHC in rat urine. A rapid and sensitive method with high reproducibility for the determination of free, conjugated and total alpha-CEHC is then established.

We previously showed that dietary sesame seed and its lignan inhibited gamma-tocopherol metabolism to 2,7,8-trimethyl-2(2'-carboxyethyl)-6-hydroxychroman (gamma-CEHC), a gamma-tocopherol metabolite, and markedly elevated tissue gamma-tocopherol concentration in rats. The aim of this study was to clarify the effect of dietary sesame seed on alpha-tocopherol metabolism. Vitamin E-deficient rats fed a vitamin E-free diet for 4 wk were fed a diet containing alpha-tocopherol, alpha- and gamma-tocopherol, or alpha-tocopherol with sesame seed for 7 d. Urinary excretion of 2,5,7,8-tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), a alpha-tocopherol metabolite, in rats fed alpha-tocopherol with sesame seed was inhibited (p<0.05) as compared with that in rats fed alpha-tocopherol alone, or alpha- and gamma-tocopherol. The gamma-CEHC excretion was also less (p<0.05) in rats fed alpha-tocopherol with sesame seed than that in rats fed alpha- and gamma-tocopherol. The inhibition of alpha- and gamma-CEHC excretion by sesame seed was accompanied by elevation (p<0.05) of the alpha- and gamma-tocopherol concentration in the liver. These results suggest that dietary sesame seed inhibits not only gamma-tocopherol metabolism to gamma-CEHC but also alpha-tocopherol metabolism to alpha-CEHC in rats.

Gamma tocopherol (gamma-T) is a recognized peroxynitrite scavenger, reputedly metabolized via the cytochrome P450 3A4 (CYP3A4). In this study, we assessed whether equipotent LDL-lowering doses of statins with or without inhibitory activity on CYP3A4 differently affect gamma-T metabolism. Patients with ATP III criteria for statin use (n=35) were randomly allocated to treatment with simvastatin 20mg/day or pravastatin 40 mg/day. Plasma lipids, alpha-tocopherol (alpha-T), gamma-T as well as the urinary excretion of the gamma-T metabolite 2,7,8-trimethyl-2-(2'carboxyethyl)-6-hydroxychroman (gamma-CEHC), were determined at baseline and after 6 weeks of treatment. Pravastatin and simvastatin equally reduced LDL-C (-42.8+/-2.9 and -42.1+/-3.0%) and alpha-T levels (-17.5+/-4.2 and -12.2+/-4.1%), and increased the alpha-T/LDL-C ratios (51.4+/-14.6 and 60.4+/-15%). Conversely, pravastatin did not affect whereas simvastatin significantly augmented plasma gamma-T levels (22+/-7.9%, p=0.009, between groups p=0.0045). Moreover, the gamma-T/LDL-C ratio increased significantly more with simvastatin than with pravastatin (124+/-23 versus 61.3+/-22.1%, p=0.05 between groups). In addition, pravastatin but not simvastatin increased the urinary excretion of gamma-CEHC (34.3+/-17.3%, p=0.056; between groups p=0.046). In conclusion, simvastatin and pravastatin produced distinct effects on gamma-T metabolism, presumably as a result of different statin-CYP interactions.

In this study, the CYP3A inducer pregnenolone-16alpha-carbonitrile (PCN) and the CYP3A inhibitor ketoconazole (KCZ) were used to investigate whether the metabolism of alpha-tocopherol to its metabolite, alpha-carboxyethyl hydroxychroman (alpha-CEHC), is CYP3A-dependent in rats. In experiment 1, two groups of Wistar rats were fed for 3 wk with either a basal diet (containing 50 ppm of alpha-tocopherol) or the same diet containing 10-fold more alpha-tocopherol. In the last 3 days, each group was divided into 2 subgroups which were given a single i.p. injection of either PCN at 75 mg/kg/d (P50 & P500 groups) or DMSO (D50 & D500 groups). The liver TBARS concentration was highest in the P50 group. Two-way ANOVA analysis showed that alpha-tocopherol levels in the plasma and liver were both significantly decreased by PCN (p < 0.0001), as were alpha-CEHC levels in the urine (p = 0.0004). In experiment 2, alpha-tocopherol levels in the liver were increased and alpha-CEHC excretion in the urine decreased in the Wistar rats fed with KCZ containing diet. In experiment 3, Wistar rats administered with dexamethasone (DEX) significantly decreased alpha-tocopherol levels in the plasma and liver and alpha-CEHC levels in the urine. These data showed CYP3A is not a major contributor of the metabolism of alpha-tocopherol to alpha-CEHC. Nevertheless, vitamin E status was markedly reduced by CYP3A inducers due to increased lipid peroxidation and this would increase the consumption of alpha-tocopherol in the liver.

We have compared the biokinetics of deuterated natural (RRR) and synthetic (all rac) alpha-tocopherol in male apoE4-carrying smokers and nonsmokers. In a randomized, crossover study subjects underwent two 4-week treatments (400 mg/day) with undeuterated RRR- and all rac-alpha-tocopheryl acetate around a 12-week washout. Before and after each supplementation period subjects underwent a biokinetic protocol (48 h) with 150 mg deuterated RRR- or all rac-alpha-tocopheryl acetate. During the biokinetic protocols, the elimination of endogenous plasma alpha-tocopherol was significantly faster in smokers (P < 0.05). However, smokers had a lower uptake of deuterated RRR than nonsmokers, but there was no difference in uptake of deuterated all rac. The supplementation regimes significantly raised plasma alpha-tocopherol (P < 0.001) with no differences in response between smokers and nonsmokers or between alpha-tocopherol forms. Smokers had significantly lower excretion of alpha-carboxyethyl-hydroxychroman than nonsmokers following supplementation (P < 0.05). Nonsmokers excreted more alpha-carboxyethyl-hydroxychroman following RRR than all rac; however, smokers did not differ in excretion between forms. At baseline, smokers had significantly lower ascorbate (P < 0.01) and higher F(2)-isoprostanes (P < 0.05). F(2)-isoprostanes in smokers remained unchanged during the study, but increased in nonsmokers following alpha-tocopherol supplementation. These data suggest that apoE4-carrying smokers and nonsmokers differ in their handling of natural and synthetic alpha-tocopherol.

Background: Some studies have shown potential benefit of vitamin E on platelet function, but several clinical trials failed to show improved cardiovascular outcome with alpha-tocopherol supplementation. Gamma-tocopherol, a major dietary form of vitamin E, may have protective properties different from those of alpha-tocopherol., Objective: We compared the effects of supplementation with alpha-tocopherol (500 mg) and a gamma-tocopherol-rich compound (500 mg, containing 60% gamma-tocopherol) on serum and cellular tocopherol concentrations, urinary tocopherol metabolite excretion, and in vivo platelet activation in subjects with type 2 diabetes., Design: Fifty-eight subjects were randomly assigned to receive either 500 mg alpha-tocopherol/d, 500 mg mixed tocopherols/d, or matching placebo. Serum, erythrocyte, and platelet tocopherol and urinary metabolite concentrations were measured at baseline and after the 6-wk intervention. Soluble CD40 ligand, urinary 11-dehydro-thromboxane B2, serum thromboxane B2, soluble P-selectin, and von Willebrand factor were measured as biomarkers of in vivo platelet activation., Results: Serum alpha-tocopherol increased with both tocopherol treatments. Serum and cellular gamma-tocopherol increased 4-fold (P < 0.001) in the mixed tocopherol group, whereas red blood cell gamma-tocopherol decreased significantly after alpha-tocopherol supplementation. Excretion of alpha-carboxyethyl-hydroxychroman increased significantly after supplementation with alpha-tocopherol and mixed tocopherols. Excretion of gamma-carboxyethyl-hydroxychroman increased significantly after supplementation with mixed tocopherols and after that with alpha-tocopherol, which may reflect the displacement of gamma-tocopherol by alpha-tocopherol due to incorporation of the latter into lipoproteins in the liver. Neither treatment had any significant effect on markers of platelet activation., Conclusions: Supplementation with alpha-tocopherol decreased red blood cell gamma-tocopherol, whereas mixed tocopherols increased both serum alpha-tocopherol and serum and cellular gamma-tocopherol. Changes in serum tocopherol closely reflect changes in cellular concentrations of tocopherols after supplementation.

Gamma-tocopherol (gammaT) is one of the major forms of vitamin E consumed in the diet. Previous reports have suggested increased levels of nitrated gamma-tocopherol (5-NO2-gammaT) in smokers and individuals with conditions associated with elevated nitrative stress. The monitoring of 5-NO2-gammaT and its possible metabolite(s) may be a useful marker of reactive nitrogen species generation in vivo. The major pathway for the metabolism of gammaT is the cytochrome P450 dependent oxidation to its water-soluble metabolite gamma-CEHC, which is excreted in urine. In order to determine if 5-NO2-gammaT could be metabolised via the same route and detected in urine we developed a sensitive gas chromatography-mass spectrometry assay for 5-NO2-gamma-CEHC. 5-NO2-gamma-CEHC was synthesised and its structure confirmed by proton nuclear magnetic resonance and mass spectrometry. While gamma-CEHC was abundant in urine from healthy volunteers, as well as patients with coronary heart disease and type 2 diabetes, 5-NO2-gamma-CEHC was undetectable (limit of detection of 5 nM). To understand this observation we examined the uptake and metabolism of gammaT and 5-NO2-gammaT by HepG2 cells. gammaT was readily incorporated into cells and metabolised to gamma-CEHC over a period of 48 hours. In contrast, 5-NO2-gammaT was poorly incorporated into HepG2 cells and not metabolised to 5-NO2-gamma-CEHC over the same time period. We conclude that nitration of gammaT prevents its incorporation into liver cells and therefore its metabolism to the water-soluble metabolite. Whether 5-NO2-gammaT could be metabolised via other pathways in vivo requires further investigation.

We hypothesized that human plasma alpha- and gamma-tocopherol concentrations reflect differences in their kinetics, especially influenced by gamma-tocopherol metabolism. Vitamin E kinetics were evaluated in humans (n=14) using approximately 50 mg each of an equimolar ratio of d6-alpha- and d2-gamma-tocopheryl acetates administered orally. Mass spectrometry was used to measure deuterated plasma tocopherols, as well as plasma and urinary vitamin E metabolites, alpha- and gamma-carboxyethylhydroxychromans (CEHCs). Plasma d2-gamma-tocopherol fractional disappearance rates (FDR; 1.39+/-0.44 pools/day, mean+/-SD) were more than three times greater than those of d6-alpha-tocopherol (0.33+/-0.11, p<0.001). The d2-gamma-tocopherol half-life was 13+/-4 h compared with 57+/-19 for d6-alpha-tocopherol. Whereas neither plasma nor urinary d6-alpha-CEHC was detectable (limit of detection 1 nmol/L), gamma-CEHC (labeled plus unlabeled) increased from 129+/-20 to 258+/-40 nmol/L by 12 h and returned to baseline by 48 h; at 12 h d2-gamma-CEHC represented 54+/-4% of plasma gamma-CEHC. Women compared with men had a greater d2-gamma-tocopherol FDR (p<0.004) and a greater maximal plasma d2-gamma-CEHC concentration (p<0.02) and CEHC FDR (p<0.007), as well as excreting four times as much d2-gamma-CEHC (p<0.04) in urine. Thus, gamma-tocopherol is rapidly metabolized to gamma-CEHC, and to a greater degree in women than in men, whereas alpha-tocopherol is maintained in the plasma and little is metabolized to alpha-CEHC.

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.

Cigarette smoking (CS) is a well-described oxidant burden in humans. We hypothesized that CS would accelerate alpha-tocopherol (alpha-T) utilization leaving less for metabolite (CEHC) production. After labeled alpha-T consumption (75 mg each of d(3)-RRR-alpha-TAc and d(6)-all-rac-alpha-TAc) by smokers and nonsmokers (n = 10/group), CS increased alpha-T disappearance and decreased plasma and urinary CEHCs. Plasma d(3)/d(6)-alpha-T ratios were approximately 1.4 during supplementation and approximately 2 from days 5 to 17. d(3)/d(6)-alpha-CEHC ratios were on average 0.29 +/- 0.05, confirming that all-rac-alpha-tocopherol is metabolized more efficiently. CEHC may be a good marker of vitamin E status, and smokers may have an increased vitamin E requirement.

Sesame seed and oil consumption previously increased human plasma gamma-tocopherol (gamma-T) concentrations. This was attributed to the sesame lignans sesamin and sesamolin. Here, we studied the inhibition of vitamin E metabolism by a single dose of sesame oil lignans coingested with deuterated alpha- and gamma-tocopherols in human volunteers. The urinary excretion of gamma-T metabolites was significantly lower in sesame oil treated than in control subjects. Concentrations of tocopherols in blood were not affected by the treatment. In conclusion, a single dose of sesame oil, containing 136 mg sesame lignans (sesamin and sesamolin), reduces the urinary excretion of co-administered gamma-T in humans.

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.

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&#95;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.

Endogenous natriuretic factors are believed to be responsible for extracellular fluid homeostasis in mammals. A new endogenous natriuretic factor, Loma Linda University-alpha (LLU-alpha) has recently been proven to be a 2,7,8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman (gamma-CEHC), which is a metabolite of gamma-tocopherol (gamma-Toc). The purpose of this study was to investigate whether gamma-Toc could accelerate sodium excretion into rat urine as a natriuretic hormone precursor. Male SD strain rats were divided into two groups; one was a control diet group, while the other was a high NaCl group (50 g/kg diet). Next, the two groups were each subdivided into two groups consisting of a placebo group and a gamma-Toc group. After the oral administration of one experimental dose of 20 mg gamma-Toc or placebo, rat urine was collected at 6 h intervals for 24 h, and then the urine volume, sodium and potassium and gamma-CEHC content were determined. gamma-Toc increased in the urine volume of the high-NaCl intake group. The sodium excretion in the high-NaCl group given gamma-Toc was 8.29+/-2.20 g, while in the control group given gamma-Toc it was 6.24+/-1.49 g from 12-18 h. In contrast, the potassium excretion in the rat urine did not change in any of the groups. Our findings suggested that gamma-Toc accelerates the degree of sodium excretion in rats with a high sodium intake.

Cigarette smoking is associated with increased oxidative stress and increased risk of degenerative disease. As the major lipophilic antioxidant, requirements for vitamin E may be higher in smokers due to increased utilisation. In this observational study we have compared vitamin E status in smokers and non-smokers using a holistic approach by measuring plasma, erythrocyte, lymphocyte and platelet alpha- and gamma-tocopherol, as well as the specific urinary vitamin E metabolites alpha- and gamma-carboxyethyl-hydroxychroman (CEHC). Fifteen smokers (average age 27 years, smoking time 7.5 years) and non-smokers of comparable age, gender and body mass index (BMI) were recruited. Subjects completed a 7-day food diary and on the final day they provided a 24 h urine collection and a 20 ml blood sample for measurement of urinary vitamin E metabolites and total vitamin E in blood components, respectively. No significant differences were found between plasma and erythrocyte alpha- and gamma-tocopherol in smokers and non-smokers. However, smokers had significantly lower alpha-tocopherol (mean+/-SD, 1.34+/-0.31 micromol/g protein compared with 1.94+/-0.54, P = 0.001) and gamma-tocopherol (0.19+/-0.04 micromol/g protein compared with 0.26+/-0.08, P = 0.026) levels in their lymphocytes, as well as significantly lower alpha-tocopherol levels in platelets (1.09+/-0.49 micromol/g protein compared with 1.60+/-0.55, P = 0.014; gamma-tocopherol levels were similar). Interestingly smokers also had significantly higher excretion of the urinary gamma-tocopherol metabolite, gamma-CEHC (0.49+/-0.25mg/g creatinine compared with 0.32+/-0.16, P = 0.036) compared to non-smokers, while their alpha-CEHC (metabolite of alpha-tocopherol) levels were similar. There was no significant difference between plasma ascorbate, urate and F2-isoprostane levels. Therefore in this population of cigarette smokers (mean age 27 years, mean smoking duration 7.5 years), alterations to vitamin E status can be observed even without the more characteristic changes to ascorbate and F2-isoprostanes. We suggest that the measurement of lymphocyte and platelet vitamin E may represent a valuable biomarker of vitamin E status in relation to oxidative stress conditions.

2,7,8-Trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), a metabolite of gamma-tocopherol and gamma-tocotrienol, was identified as a new endogenous natriuretic factor. However, gamma-tocopherol and gamma-tocotrienol, both precursors of gamma-CEHC, have never directly been observed to have natriuretic potency. Thus, we investigated whether gamma-tocotrienol could cause natriuresis and diuresis in rats. The rats were divided into two groups that were given a control or a high-sodium diet for 4 weeks, and then subdivided into placebo and gamma-tocotrienol subgroups given only corn oil-removed vitamin E and oil supplemented with gamma-tocotrienol, respectively. After oral administration of three experimental doses, rat urine was collected and gamma-CEHC, urine volume, sodium, and potassium content were determined. Only in rats given a high-NaCl diet did gamma-tocotrienol accelerate and increase sodium excretion, showing no effect on potassium excretion. Sodium excretion in the high-NaCl group given gamma-tocotrienol was 5.06 +/- 2.70 g/day, and in the control group given gamma-tocotrienol, 0.11 +/- 0.06 g/day. Furthermore, gamma-tocotrienol affected urine volume in the specific condition of high-NaCl body stores and gamma-tocotrienol supplementation. In this study, we found that gamma-tocotrienol, one of the natural vitamin E homologs, stimulates sodium excretion in vivo, suggesting that gamma-tocotrienol possesses a hormone-like natriuretic function.

Approximately one-third to one-half of individuals harbor the colonic bacteria that are capable of metabolizing the soy isoflavone daidzein to equol. Results of prior studies suggest beneficial effects of producing equol in relation to breast cancer risk, potentially through effects on endogenous hormones. High urinary excretion of 2-hydroxyestrone (2-OH E(1)) relative to 16alpha-hydroxyestrone (16alpha-OH E(1)) has been associated with a reduced risk of breast cancer. In this pilot study we examined associations between urinary excretion of equol and 2-OH E(1), 16alpha-OH E(1), and their ratio, and investigated whether excretion of these estrogen metabolites differed between two samples collected 48h apart. Isoflavones (genistein, daidzein, O-desmethylangolensin (ODMA), and equol) were measured in two overnight urines from 126 women. Excretion of 2-OH E(1) and 16alpha-OH E(1) were measured in the first overnight urine from all 126 women and in the second overnight urine from 30 of these women; there were no significant differences between samples collected 48h apart in excretion of 2-OH E(1) or 16alpha-OH E(1) (P=0.75 and 0.17, respectively). Among all women, correlations between total isoflavone excretion (sum of genistein, daidzein, ODMA, and equol) and estrogen metabolites were non-significant (P>0.05). Among women with detectable levels of equol, total isoflavone excretion was significantly positively correlated with 16alpha-OH E(1) (r=0.32, P=0.02), but was not correlated with 2-OH E(1) or 2-OH E(1):16alpha-OH E(1) ratio (r=0.21, P=0.14, and r=-0.05, P=0.70, respectively). Equol excretion (adjusted for other isoflavone excretion) was significantly positively correlated with 2-OH E(1):16alpha-OH E(1) ratio (r=0.38, P=0.005), but was not correlated with 2-OH E(1) or 16alpha-OH E(1) (r=0.15, P=0.29, and r=-0.17, P=0.24, respectively). The finding that equol excretion, but not total isoflavone excretion, correlated positively with the 2-OH E(1):16alpha-OH E(1) ratio suggests that the colonic bacterial profile associated with equol production may be involved in estrogen metabolism, and may therefore possibly influence breast cancer risk.

We have investigated the metabolism of isoflavones and lignans in germ-free (GF) rats and rats associated with human faecal bacteria (human flora associated [HFA] rats), in order to provide unequivocal evidence for the role of the gut microflora in the absorption and metabolism of these phytoestrogens. Furthermore, we have investigated whether certain metabolic characteristics (high equol-producing and low equol-producing status) of human intestinal floras can be transferred to GF rats. Germ-free rats fed a soy-isoflavone containing diet excreted large quantities of daidzein and genistein in urine indicating that the gut microflora is not required for the absorption of isoflavones. The isoflavone metabolites equol, O-desmethylangolensin and the lignan enterolactone were not detectable in urine from the GF rats, but were present in HFA rat urine, indicating that they were products of gut microflora activity. Colonization of GF rats with a faecal flora from a human subject with the capacity to convert daidzein to equol, resulted in the rats excreting substantial amounts of the metabolite. In contrast, equol was undetectable in urine of HFA rats associated with a faecal flora from a low equol-producing subject. The results therefore show that the inability of some subjects to produce equol is a consequence of the lack of specific components of the gut microflora.

We present a method for the determination of the isoflavan equol in plasma and urine. This estrogenic isoflavan, which is formed by the action of the intestinal microflora, may have higher biological activity than its precursor daidzein. High urinary excretion of equol has been suggested to be associated with a reduction in breast cancer risk. The method is based on time-resolved fluoroimmunoassay, using a europium chelate as a label. After synthesis of 4'-O-carboxymethylequol the compound is coupled to bovine serum albumin (BSA), then used as antigen to immunize rabbits. The tracer with the europium chelate is synthesized using the same 4'-O-derivative of equol. After enzymatic hydrolysis (urine) or enzymatic hydrolysis and ether extraction (plasma) the immunoassay is carried out. The antiserum cross-reacted to variable extent with some isoflavonoids. For the plasma method the cross-reactivity does not seem to influence the results, which were highly specific. The overestimation of the values using the urine method (164%) compared to the results obtained by a gas chromatography-mass spectrometry (GC-MS) method is probably due to some influence of the matrix on the signal, and interference of structurally related compounds. It is suggested that plasma assays are used but if urine samples are measured a formula has to be used to correct the values making them comparable to the GC-MS results. The correlation coefficients between the time-resolved fluoroimmunoassay (TR-FIA) methods and GC-MS methods were high; r-values for the plasma and urine method, were 0.98 and 0.91, respectively. The intra-assay coefficient of variation (CV%) for the TR-FIA plasma and urine results at three different concentrations vary between 5.5-6.5 and 3.4-6.9, respectively. The inter-assay CV% varies between 5.4-9.7 and 7.4-7.7, respectively. The working ranges of the plasma and urine assay are 1.27-512 and 1.9-512nmol/l, respectively.

Method: alpha-Tocopherol and gamma-tocopherol are metabolized into 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC) and 2,7,8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman (gamma-CEHC), respectively. We analyzed alpha- and gamma-CEHC concentrations in human serum and urine by high-performance liquid chromatography during administration of alpha-tocopherol. Fourteen healthy adult male volunteers received 1,200 IU per day of RRR-alpha-tocopherol for 28 days. Blood and urine samples were obtained on days 0, 14, 28, and 56., Results: During alpha-tocopherol administration, the plasma gamma-tocopherol concentration decreased significantly, but there was marked elevation of the alpha-tocopherol concentration. Increased concentration of alpha-CEHC and gamma-CEHC in both serum and urine indicated the acceleration of vitamin E metabolism., Conclusion: High-dose administration of alpha-tocopherol caused an increase of gamma-tocopherol metabolism, which might have caused a decrease of the plasma gamma-tocopherol concentration.

Background: Despite significant interest in the risks and benefits of phytoestrogens to human health, few data exist on their pharmacokinetics in humans., Objective: We investigated the pharmacokinetics of the (13)C isotopic forms of daidzein and genistein in healthy humans, specifically addressing intraindividual variability, effect of increasing intake, and influence of prolonged exposure to a soy food diet., Design: Premenopausal women (n = 16) were administered 0.4 mg [(13)C]daidzein or [(13)C]genistein/kg body wt orally on 3 occasions, including once after eating soy foods for 7 d. On a further occasion the dose was doubled. Plasma and urinary [(13)C]isoflavone concentrations were measured by mass spectrometry., Results: Serum concentrations of [(13)C]genistein and [(13)C]daidzein peaked after 5.5 and 7.4 h, respectively. The systemic bioavailability and maximum serum concentration of [(13)C]genistein were significantly greater than those of [(13)C]daidzein. The bioavailability of both isoflavones did not increase linearly when the dietary intake was doubled. The mean volume of distribution normalized to bioavailability (V(d)/F), clearance rate, and half-life of [(13)C]daidzein were 336.25 L, 30.09 L/h, and 7.75 h, respectively; the corresponding values for [(13)C]genistein were 258.76 L, 21.85 L/h, and 7.77 h. The average recovery of [(13)C]daidzein and [(13)C]genistein in urine was 30.1% and 9.0% of the dose ingested, respectively., Conclusions: The serum pharmacokinetics of [(13)C]daidzein and [(13)C]genistein were reproducible among healthy women, and genistein was more bioavailable than was daidzein. Pharmacokinetics were unaffected by chronic exposure to soy foods. Urinary isoflavone concentrations correlated poorly with maximal serum concentrations, indicating the limitations of urine measurements as a predictor of systemic bioavailability. The bioavailability of both isoflavones was nonlinear at higher intakes, suggesting that uptake is rate-limiting and saturable.

Glucuronide and sulphate conjugates of 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), the major metabolite of alpha-tocopherol (vitamin E), have been synthesized and used for the first direct analysis of conjugated urinary vitamin E metabolites. The metabolites of vitamin E (alpha-tocopherol) could be useful as markers of the function(s) of vitamin E in vivo. A number of methods have been described for the analysis of urinary vitamin E metabolites but these have relied on either acid or enzymatic deconjugation of the metabolites prior to analysis by high performance liquid chromatography or gas chromatography/mass spectrometry. These methods have provided useful information about the amount and types of metabolites excreted in the urine but suffer from a number of disadvantages. Deconjugation has been shown to produce artifacts as a result of the conversion of alpha-CEHC to alpha-tocopheronolactone and the efficiency of deconjugation is also difficult to assess. Methods that allow the direct measurement of the conjugated metabolites would overcome these problems and would also substantially reduce the preparation and analysis time. Here we describe the use of conjugated standards to characterize alpha-CEHC conjugates in human urine by tandem mass spectrometry (MS-MS). The future use of MS-MS to measure urinary vitamin E metabolites is also discussed., (Copyright 2002 Elsevier Science Inc.)

Daidzein and its main metabolite equol are isoflavone phytoestrogens. Several studies have suggested that intake of an isoflavone-rich diet may prevent hormone-related cancer and estrogen-related disorders (cardiovascular disease, osteoporosis and menopausal symptoms). To better understand the role of isoflavones in preventing such severe disease, several methods have been developed to measure these compounds in biological fluids. However, the analytical procedures to measure isoflavones are often time-consuming and require highly skilled technicians. In this paper we describe a method for urinary daidzein and equol measurement that combines solid phase extraction and HPLC purification before gas chromatographic determination. The specificity of the method was confirmed by the gas chromatography-mass spectrometry technique. The mean recovery of daidzein and equol was 94.6% and 97.0%, respectively. The repeatability of the method was in the range of 2.0-7.4% for daidzein and 1.3-4.9% for equol. A linear relationship between observed and expected values was found in the dilution (r2=0.9983 for daidzein; r2=0.9982 for equol) and addition (r2=0.9984 for daidzein; r2=0.9989 for equol) assays. The method is suitable to measure changes in the urinary excretion of isoflavones and to investigate urinary isoflavonoids as biomarkers of isoflavone exposure.

We showed previously that dietary sesame seed and its lignans elevate the tocopherol concentration in rats. To clarify their effect on tocopherol metabolism, we determined in this study the urinary excretion of 2,7,8-trimethyl-2(2'-carboxyethyl)-6-hydroxychroman (gamma-CEHC), a gamma-tocopherol metabolite, in rats fed sesame seed or its lignans. Rats were fed diets with or without sesame seed for 28 d in Experiment 1, and for 1, 3 and 7 d in Experiment 2. On d 28, dietary sesame seed elevated (P < 0.05) gamma-tocopherol concentrations in liver, kidney, brain and serum, and decreased (P < 0.05) urinary excretion of gamma-CEHC. The excretion was completely inhibited by feeding sesame seed on d 1 and 3. In Experiment 3, the effects of dietary sesamin and sesaminol (major lignans in sesame seed) or ketoconazole (a selective inhibitor of cytochrome P(450) (CYP)3A on urinary excretion of gamma-CEHC in rats fed gamma-tocopherol were examined. The urinary gamma-CEHC in rats fed sesamin or sesaminol was markedly lower than in rats fed gamma-tocopherol alone (P < 0.05). Dietary ketoconazole also inhibited (P < 0.05) urinary excretion of gamma-CEHC, and elevated (P < 0.05) gamma-tocopherol concentrations in tissues and serum of rats fed gamma-tocopherol. These data suggest that sesame seed and its lignans elevate gamma-tocopherol concentration due to the inhibition of CYP3A-dependent metabolism of gamma-tocopherol.

gamma-tocopherol is the major form of vitamin E in many plant seeds and in the US diet, but has drawn little attention compared with alpha-tocopherol, the predominant form of vitamin E in tissues and the primary form in supplements. However, recent studies indicate that gamma-tocopherol may be important to human health and that it possesses unique features that distinguish it from alpha-tocopherol. gamma-Tocopherol appears to be a more effective trap for lipophilic electrophiles than is alpha-tocopherol. gamma-Tocopherol is well absorbed and accumulates to a significant degree in some human tissues; it is metabolized, however, largely to 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), which is mainly excreted in the urine. gamma-CEHC, but not the corresponding metabolite derived from alpha-tocopherol, has natriuretic activity that may be of physiologic importance. Both gamma-tocopherol and gamma-CEHC, but not alpha-tocopherol, inhibit cyclooxygenase activity and, thus, possess antiinflammatory properties. Some human and animal studies indicate that plasma concentrations of gamma-tocopherol are inversely associated with the incidence of cardiovascular disease and prostate cancer. These distinguishing features of gamma-tocopherol and its metabolite suggest that gamma-tocopherol may contribute significantly to human health in ways not recognized previously. This possibility should be further evaluated, especially considering that high doses of alpha-tocopherol deplete plasma and tissue gamma-tocopherol, in contrast with supplementation with gamma-tocopherol, which increases both. We review current information on the bioavailability, metabolism, chemistry, and nonantioxidant activities of gamma-tocopherol and epidemiologic data concerning the relation between gamma-tocopherol and cardiovascular disease and cancer.

A new, crystalline 5'-thiol modifier phosphoramidite monomer (3), suitable for DNA synthesis, has been prepared. This monomer has been built into an oligonucleotide using the standard protocol. After cleavage, purification and removal of the trityl group with Ag(+), a free 5'-thiol terminal oligonucleotide (15) has been obtained which was subsequently coupled to a cysteine derivative via a disulfide bridge to afford conjugate 16.

In this study, we investigated a change in the excretory content of 2,7,8-trimethyl-2(2'-carboxyethyl)-6-hydroxychroman (gamma-CEHC), a gamma-tocopherol (gamma-Toc) metabolite, in rat urine and bile by using a new high-performance liquid chromatography-electrochemical detection (HPLC-ECD) method. In this determination, CEHC [alpha- and gamma-CEHC, where alpha-CEHC = 2,5,7,8-tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman] in the biological specimens were treated with 3 N methanolic HCl to hydrolyze conjugates and to promote esterification. The methylated samples were extracted by n-hexane/water (1:2). The analyses of the methyl esters of alpha-CEHC and gamma-CEHC were performed by an HPLC-ECD using an ODS-3 column at 35 degrees C. The mobile phase was acetonitrile/water (45:55, vol/vol) containing 50 mM sodium perchlorate. After rat urine and bile samples, respectively, were methylated as described above, methylated biliary metabolites were identified by liquid chromatography-mass spectrometry as methyl esters of gamma-CEHC. Furthermore, we examined the differences in the excretion of gamma-CEHC between rat urine and bile after an oral administration of gamma-Toc or alpha- + gamma-Toc by the above HPLC method. In the gamma-Toc group, each vitamin E-deficient rat was given 0.5 mL of a stripped corn oil preparation containing 10 mg of gamma-Toc. In the alpha- + gamma-Toc group, the rat was given 10 mg of alpha-Toc and 10 mg of gamma-Toc. The content of gamma-CEHC in rat urine from the alpha- + gamma-Toc group was increased more in comparison to the gamma-Toc group at 18-36 h after oral administration. Moreover, the content of gamma-CEHC in rat bile in the alpha- + gamma-Toc group was increased more in comparison to the gamma-Toc group at 6-18 h after oral administration. Therefore, we have suggested that gamma-CEHC was shifted mainly to urinary excretion after gamma-CEHC had been excreted into the bile. Furthermore, we assume that alpha-Toc may affect the metabolism of gamma-Toc to gamma-CEHC in the body.

The capacity to convert the soy isoflavone daidzein to equol in vivo is presumably determined by an individual's intestinal microfloral populations; however, diet may also influence this conversion. The objectives of the present study were to determine whether a 1-mo supplementation of dietary fiber as wheat bran increases urinary equol excretion in equol excreters and stimulates equol production in nonexcreters and whether longer-term soy isoflavone intake increases equol production or alters overall urinary isoflavone excretion. First, we screened 74 women, ages 20-40 y, and determined their equol-excreter status. In these women, health and lifestyle patterns and habitual dietary intake did not differ according to equol-excreter status. Next, 26 of the women (13 equol excreters and 13 nonexcreters) were assigned (blocked on equol-excreter status) to either longer-term (1 mo) or short-term (4 d) soy protein supplementation. Within each soy treatment group, women participated in two 1-mo intervention periods (the exact length was determined by each woman's menstrual cycle) during which they consumed their usual diets supplemented daily with either 0 or 16 g dietary fiber in a randomized crossover design. A 1-mo washout period separated the two diet periods. Among the 19 women who completed both periods, fiber supplementation did not increase equol production in equol excreters or nonexcreters. In addition, isoflavonoid excretion did not differ by fiber dose or length of soy intervention. These results suggest that a daily 16 g-fiber dose as wheat bran and the addition of soy protein do not alter significantly the capacity of colonic microflora to produce equol.

Limited information is available regarding metabolism of vitamin E forms, especially the tocotrienols. Carboxyethyl-hydroxychromans (alpha- and gamma-CEHC) are human urinary metabolites of alpha- and gamma-tocopherols, respectively. To evaluate whether tocotrienols are also metabolized and excreted as urinary CEHC, urine was monitored following tocotrienol supplementation. Complete (24 h) urine collections were obtained for 2 d prior to (baseline), the day of, and 2 d after human subjects (n = 6) ingested tocotrienol supplements. The subjects consumed 125 mg gamma-tocotrienyl acetate the first week, then the next week 500 mg; then 125 mg alpha-tocotrienyl acetate was administered the third week, followed by 500 mg the fourth week. Urinary alpha- and gamma-CEHC were measured by high-performance liquid chromatography with electrochemical detection. Urinary gamma-CEHC levels rose about four- to sixfold in response to the two doses of gamma-tocotrienol and then returned to baseline the following day. Significant (P < 0.0001) increases in urinary alpha-CEHC were observed only following ingestion of 500 mg alpha-tocotrienyl acetate. Typically, 1-2% of alpha-tocotrienyl acetates or 4-6% of gamma-tocotrienyl acetates were recovered as their respective urinary CEHC metabolites. A gamma-CEHC excretion time course showed an increase in urinary gamma-CEHC at 6 h and a peak at 9 h following ingestion of 125 mg gamma-tocotrienyl acetate. In summary, tocotrienols, like tocopherols, are metabolized to CEHC; however, the quantities excreted in human urine are small in relation to dose size.

Patients with alpha-tocopherol transfer protein (alpha-TTP) defects experience neurological symptoms characteristic of vitamin E deficiency and depend on continuous high alpha-tocopherol supplements. We investigated the excretion of 2,5,7, 8-tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), a urinary metabolite of alpha-tocopherol, as a putative marker for the alpha-tocopherol status of alpha-TTP-deficient patients and control subjects. In three patients vitamin E supplementation was stopped for short periods of time, during which plasma alpha-tocopherol concentrations and urinary alpha-CEHC excretion were measured. In the patients, plasma alpha-tocopherol decreased below normal (<5 micromol/l) but alpha-CEHC excretion remained above the range of unsupplemented control subjects (0.118-0.306 mg/day, n = 6). In healthy subjects, however, alpha-CEHC excretion was increased only after surpassing a plasma alpha-tocopherol threshold of 30-40 micromol/l. Such a threshold did not exist in patients. The general mechanism of alpha-tocopherol degradation did not appear to differ between patients and control subjects. The presumed mechanism of omega- and subsequent beta-oxidation was supported by the detection of alpha- CPHC, an alpha -CEHC homolog with a side chain longer by 3 carbon atoms, both in supplemented patients and in control subjects.

There is currently interest in measuring urinary metabolites of vitamin E. It has been suggested that alpha-to-copheronolactone (alphaTL), with an oxidized chroman ring, may be an indicator of in vivo oxidative stress and 2,5,7,8-tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), with a shortened side chain but intact hydroxychroman ring, may provide a measure of adequate or excess vitamin E status. To date, methods in the literature have tended to concentrate on the estimation of single metabolites. We describe the establishment and validation of a relatively simple and reproducible method to extract and quantitate a range of vitamin E metabolites using 0.5 ml of human urine. The vitamin E metabolites were extracted from urine using solid phase extraction cartridges, deconjugated enzymatically, and analyzed using gas chromatography-mass spectrometry. Using this method we have identified alphaTL and the CEHC metabolites derived from alpha-, delta-, and gamma-tocopherol. In addition we have tentatively identified a novel group of vitamin E metabolites, which are related to the CEHCs but have three extra carbons in the side chain. The possibility of the artifactual oxidation of alpha-CEHC to alphaTL during the assay procedure is also discussed.

1. Deconjugation by sulphate transfer and intestinal absorption of troglitazone sulphate (M1), the major metabolite of a thiazolidinedione antidiabetic drug, troglitazone, were studied in the male F344 rat using 14C-troglitazone, 4C-M1 and 35S-M1. 2. Some part of M1, produced in the liver and excreted mostly in the bile, was deconjugated in the intestine to the parent compound, troglitazone, by arylsulphate sulphotransferase originated from intestinal flora. However, deconjugation of M1 was not catalyzed by arylsulphatases. Caecal injection of M1 led to the appearance of troglitazone and M1 in plasma. 3. Biliary excretion mostly as M1, and, following absorption, as M1 and troglitazone after deconjugation, were indicated as the basis for the enterohepatic circulation of troglitazone. 4. Enterohepatic circulation may prolong the pharmacological effects of troglitazone.

Objective: To determine pharmacokinetics of troglitazone in healthy cats after i.v. and oral administration of a single dose of the drug., Animals: 5 healthy ovariohysterectomized adult cats., Procedure: Using a randomized crossover design, cats were given 5 mg of troglitazone/kg of body weight i.v. and 40 mg of troglitazone/kg orally. Blood and urine samples were collected after drug administration, and concentrations of troglitazone in plasma and urine were determined by use of high-performance liquid chromatography., Results: Area-moment analysis was used to calculate pharmacokinetic variables. Terminal phase half-life was 1.1 +/- 0.1 hours. Steady-state volume of distribution was 0.23 +/- 0.15 L/kg. After i.v. administration, clearance was 0.33 +/- 0.04 L/h/kg. Drug was not detected in urine samples. Mean bioavailability of orally administered troglitazone was 6.9%., Conclusions and Clinical Relevance: The overall disposition of troglitazone in cats was similar to that reported in other species, including humans. Troglitazone has low and variable oral bioavailability. Clearance of the compound is moderate. Little if any unchanged troglitazone is excreted in urine; thus, metabolism and biliary excretion play predominant roles in elimination of the drug. On the basis of troglitazone pharmacokinetics in healthy cats, as well as on the basis of pharmacodynamics of the drug in humans and other animals, a regimen that uses a dosage of 20 to 40 mg/kg administered orally once or twice per day to cats will produce plasma concentrations of the insulin-sensitizing agent that have been documented to be effective in humans.

The in vivo estrogenic potential of the flavonoids apigenin, kaempferol, genistein and equol was investigated in immature female mice. Genistein and equol, administered by gavage for 4 consecutive days [post-natal day (PND) 17-20, 100 mg/kg body weight], was found to significantly increase uterine weights and the overall uterine concentration of estrogen receptor alpha (ERalpha). In kaempferol- and equol-exposed mice the cytosolic ERalpha concentration was significantly increased as compared to the solvent control, which is speculated to result in an increased sensitivity of the uterus to subsequently encountered estrogens. Oral administration of equol, genistein, biochanin A and daidzein to 6-week-old female mice revealed a great variation in their systemic bioavailability. The urinary recovery of equol was thus over 90% of a single gavage administered dose, whereas the urinary recoveries of biochanin A, genistein and daidzein were 16, 11 and 3%, respectively. Most of the metabolites were either hydroxylated or dehydrogenated forms of the parent compounds. The in vitro estrogenic potency of some of the metabolites was greater than that of the parent compounds, whereas others were of similar or lower potency. Bioavailability, metabolism, the ability to alter ERalpha distribution in the uterus and the estrogenic potential of parent compound and metabolites may thus contribute to the differences in in vivo estrogenicity of dietary flavonoids.

Increased urinary excretion of equol, a metabolite of the isoflavone daidzein, has been associated with a reduced risk of breast cancer. This risk reduction has generally been presumed to be a consequence of increased isoflavone consumption. However, only 30-40% of the population excretes more than trace amounts of equol, regardless of isoflavone intake. Accordingly, we hypothesized that the observed apparent protective effect of equol is at least in part attributable to hormonal differences between equol excretors and non-excretors, and that these differences are largely independent of isoflavone intake. We measured plasma hormone and sex hormone binding globulin (SHBG) concentrations in 14 normally cycling premenopausal women during each of three diet periods in which they consumed differing isoflavone doses (0.15, 1.0, and 2.0 mg/kg of body weight/day) as a component of soy protein isolate. The plasma hormone and SHBG concentrations of equol excretors (n = 5) were then compared with those of the non-excretors (n = 9). Results showed that even at the lowest dose, urinary equol excretion values for excretors far exceeded those for non-excretors consuming the highest dose. At all doses, equol excretors generally had lower concentrations of estrone, estrone-sulfate, testosterone, androstenedione, dehydroepiandrosterone (DHEA), DHEA-sulfate, and cortisol and higher concentrations of SHBG and midluteal progesterone, a hormonal pattern overall consistent with lowered breast cancer risk. In conclusion, the association of equol excretion and lowered breast cancer risk may largely reflect the tendency of equol excretors to have more favorable hormonal profiles, as opposed to merely reflecting increased isoflavone intake. Equol may be a marker for the presence of colonic bacterial enzymatic activity that increases fecal steroid excretion. Alternatively, equol itself, even with very modest isoflavone intake, may exert beneficial effects on the regulation of endogenous hormones.