Infant Rhesus Macaque Brain α-Tocopherol Stereoisomer Profile Is Differentially Impacted by the Source of α-Tocopherol in Infant Formula.

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.

Metabolic syndrome increases dietary α-tocopherol requirements as assessed using urinary and plasma vitamin E catabolites: a double-blind, crossover clinical trial.

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 (d6-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 (d6)-α-CEHC (0.04 ± 0.02 compared with 0.13 ± 0.02 µmol/g creatinine, respectively; P = 0.002), and 58% less d6-α-CMBHC (0.017 ± 0.004 compared with 0.041 ± 0.004 µmol/g creatinine, respectively; P = 0.0009) and had 52% lower plasma d6-α-CEHC areas under the concentration curves [area under the curve from 0 to 24 h (AUC0-24h): 27.7 ± 7.9 compared with 58.4 ± 7.9 nmol/L × h, respectively; P = 0.01]. d6-α-CEHC peaked before d6-α-T in 77 of 80 paired plasma concentration curves. Urinary d6-α-CEHC 24-h concentrations were associated with the plasma AUC0-24 h of d6-α-T (r = 0.53, P = 0.02) and d6-α-CEHC (r = 0.72, P = 0.0003), and with urinary d6-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.

Ionic liquid-molecularly imprinted polymers for pipette tip solid-phase extraction of (Z)-3-(chloromethylene)-6-flourothiochroman-4-one in urine.

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).

γ-Tocopherol-rich supplementation additively improves vascular endothelial function during smoking cessation.

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.

Toxicogenomics and metabolomics of pentamethylchromanol (PMCol)-induced hepatotoxicity.

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.

Daily consumption of an aqueous green tea extract supplement does not impair liver function or alter cardiovascular disease risk biomarkers in healthy men.

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.

Cigarette smokers differ in their handling of natural (RRR) and synthetic (all rac) alpha-tocopherol: a biokinetic study in apoE4 male subjects.

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.

Nitration of gamma-tocopherol prevents its oxidative metabolism by HepG2 cells.

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.

{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.

Cigarette smokers have decreased lymphocyte and platelet alpha-tocopherol levels and increased excretion of the gamma-tocopherol metabolite gamma-carboxyethyl-hydroxychroman (gamma-CEHC).

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.

Cigarette smoking increases human vitamin E requirements as estimated by plasma deuterium-labeled CEHC.

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.

Consumption of sesame oil muffins decreases the urinary excretion of gamma-tocopherol metabolites in humans.

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.

Urinary equol excretion in relation to 2-hydroxyestrone and 16alpha-hydroxyestrone concentrations: an observational study of young to middle-aged women.

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.

Time-resolved fluoroimmunoassay for equol in plasma and urine.

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.

Quantitative analysis of urinary daidzein and equol by gas chromatography after solid-phase extraction and high-performance liquid chromatography.

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.

gamma-tocopherol, the major form of vitamin E in the US diet, deserves more attention.

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.

Synthesis and application of a novel, crystalline phosphoramidite monomer with thiol terminus, suitable for the synthesis of DNA conjugates.

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.

Estrogenic activity of flavonoids in mice. The importance of estrogen receptor distribution, metabolism and bioavailability.

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.

Premenopausal equol excretors show plasma hormone profiles associated with lowered risk of breast cancer.

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.

Interindividual variation in metabolism of soy isoflavones and lignans: influence of habitual diet on equol production by the gut microflora.

The soy isoflavones, daidzein and genistein, and the lignans, matairesinol and secoisolariciresinol, are phytoestrogens metabolized extensively by the intestinal microflora. Considerable important evidence is already available that shows extensive interindividual variation in isoflavone metabolism, and we have investigated the extent of this variation in a crossover study of a soy-containing food low or high in isoflavones (each treatment period lasted for 17 days, and the 2 treatment periods were separated by a 25-day washout period) in 24 healthy subjects [19 women and 5 men, mean age 30 yr, range 19-40, mean body mass index 22.5 +/- 3.5 (SD) kg/m2]. There was a 16-fold variation in total isoflavonoid excretion in urine after the high-isoflavone treatment period. The variation in urinary equol excretion was greatest (664-fold), and subjects fell into two groups: poor equol excretors and good equol excretors (36%). A significant negative correlation was found between the proportion of energy from fat in the habitual diet and urinary equol excretion (r = -0.55; p = 0.012). Good equol excretors consumed less fat as percentage of energy than poor excretors (26 +/- 2.3% compared with 35 +/- 1.6%, p < 0.01) and more carbohydrate as percentage of energy than poor excretors (55 +/- 2.9% compared with 47 +/- 1.7%, p < 0.05). Interindividual variation in the urinary excretion of O-desmethyl-angolensin (O-DMA) was also apparent (76-fold after the high-isoflavone treatment period), but there was no relationship between equol excretion and O-DMA excretion. Enterolactone was the major lignan metabolite in urine and plasma but showed less interindividual variation than equol and O-DMA. It is suggested that the dietary fat intake decreases the capacity of gut microbial flora to synthesize equol.