Correlations between urinary concentrations and dietary intakes of flavonols in the European Prospective Investigation into Cancer and Nutrition (EPIC) study.

PURPOSE: In this study, we aimed to study the correlation between acute and habitual intakes of flavonols, their main food sources and their 24-h urinary concentrations in an European population. METHODS: A 24-h dietary recall (24-HDR) and 24-h urine samples were collected on the same day from a convenience subsample of 475 men and women from four countries (France, Italy, Greece and Germany) of the European Prospective Investigation into Cancer and Nutrition (EPIC) study. A standardized 24-HDR software and a country/centre-specific validated dietary questionnaire (DQ) were used to collect acute and habitual dietary data, respectively. The intake of dietary flavonols was estimated using the Phenol-Explorer database. Urinary flavonols (quercetin, isorhamnetin, and kaempferol) were analysed using tandem mass spectrometry with a previous enzymatic hydrolysis. RESULTS: Weak partial Spearman correlations between both dietary acute and habitual intake and urinary concentrations of quercetin (both Rpartial ~ 0.3) and total flavonols (both Rpartial ~ 0.2) were observed. No significant correlations were found for kaempferol and isorhamentin. Regarding flavonol-rich foods, weak correlations were found between urinary concentrations of quercetin and total flavonols and the acute intake of onions and garlics, fruits, tea, and herbal tea (all Rpartial ~ 0.2). For habitual intake, statistically significant correlations were only found between urinary quercetin concentration and herbal tea (Rpartial = 0.345) and between urinary total flavonol concentration and tea, and herbal tea consumption (Rpartial ~ 0.2). CONCLUSIONS: Our results suggest that urinary quercetin level can be used as potential concentration biomarkers of both acute and habitual quercetin intake, while urinary concentrations of flavonols are unlikely to be useful biomarkers of individual flavonol-rich foods.

Flavanol Bioavailability in Two Cocoa Products with Different Phenolic Content. A Comparative Study in Humans.

Cocoa has beneficial health effects partly due to its high flavanol content. This study was aimed at assessing the absorption and metabolism of polyphenols in two soluble cocoa products: a conventional (CC) and a flavanol-rich product (CC-PP). A crossover, randomized, blind study was performed in 13 healthy men and women. On two different days, after an overnight fast, volunteers consumed one serving of CC (15 g) or CC-PP (25 g) in 200 mL of semi-skimmed milk containing 19.80 mg and 68.25 mg of flavanols, respectively. Blood and urine samples were taken, before and after CC and CC-PP consumption, and analyzed by high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometry (HPLC-ESI-QToF-MS). Up to 10 and 30 metabolites were identified in plasma and urine, respectively. Phase II derivatives of epicatechin were identified with kinetics compatible with small intestine absorption, although the most abundant groups of metabolites were phase II derivatives of phenyl-γ-valerolactone and phenylvaleric acid, formed at colonic level. 5-(4'-Hydroxyphenyl)-γ-valerolactone-sulfate could be a sensitive biomarker of cocoa flavanol intake. CC and CC-PP flavanols showed a dose-dependent absorption with a recovery of 35%. In conclusion, cocoa flavanols are moderately bioavailable and extensively metabolized, mainly by the colonic microbiota.

Association between different dietary polyphenol subclasses and the improvement in cardiometabolic risk factors: evidence from a randomized controlled clinical trial.

AIMS: Due to their different chemical structures and metabolism, polyphenol subclasses may have specific impact on cardiometabolic risk factors. Our aim was to evaluate whether the intake of different polyphenol subclasses is associated with clinical outcomes beneficially improved by polyphenols in a nutritional trial performed by our group (postprandial lipid response, glucose homeostasis, early insulin secretion and oxidative stress). METHODS: The present study is a secondary analysis of a nutritional intervention study with a diet naturally rich in polyphenols. The data are derived from 78 participants at high cardiovascular risk who completed the ETHERPATH trial. The associations between variations in polyphenol subclasses (phenolic acids, anthocyanidins, flavones, flavan-3-ols, flavonols and flavanones) and clinical outcomes beneficially influenced by polyphenols were firstly explored by Spearman's correlation. Thereafter, adjustment for gender, age and body mass index (BMI) was run. Linear regression analysis was used to assess the class of polyphenols that best predicted the outcome. RESULTS: Flavanone intake was inversely correlated with postprandial lipid response, whereas flavone intake was related to postchallenge glucose response. Anthocyanidins and flavan-3-ols associated positively with early insulin secretion. The decrease in urinary isoprostanes correlated with anthocyanidins, flavan-3-ols and flavonols. Correlations did not change after adjustment for gender, age, and BMI. Linear regression analysis showed an independent association between flavonols and urinary isoprostanes, whereas early insulin secretion was mainly associated with flavan-3-ols intake. CONCLUSIONS: The results of this study show that a polyphenol-rich diet may have a pleiotropic effect on cardiometabolic risk factors thanks to the specific action of different polyphenol subclasses.

Tissue Distribution, Excretion, and Metabolic Profile of Dihydromyricetin, a Flavonoid from Vine Tea (Ampelopsis grossedentata) after Oral Administration in Rats.

Dihydromyricetin (DMY), a flavanonol compound found as the most abundant and bioactive constituent in vine tea (Ampelopsis grossedentata), possesses numerous biological activities. In the present study, an HPLC-MS/MS method for the determination of DMY in tissues, urine, and feces was developed and applied to the tissue distribution and excretion study after oral administration in rats, and the metabolic profile of DMY was further investigated using UPLC-QTOF-MS. The results indicated that DMY could be distributed rapidly in various tissues and highly in the gastrointestinal tract. The elimination of DMY occurred rapidly as well, and most unconverted forms were excreted in feces. A total of eight metabolites were identified in urine and feces, while metabolites were barely found in plasma. The predicted metabolic pathways including reduction, dehydroxylation, methylation, glucuronidation, and sulfation were proposed. The present findings may provide the theoretical basis for evaluating the biological activities of DMY and will be helpful for its future development and application.

Identification of 3',4'-Dimethoxy Flavonol-3-ß-d-Glucopyranoside Metabolites in Rats by Liquid Chromatography-Electrospray Ionization Ion Trap Mass Spectrometry.

A method using liquid chromatography-electrospray ionization ion trap mass spectrometry was established for the identification of metabolites in feces, urine and bile in rats after oral administration of 3',4'-dimethoxy flavonol-3-ß-d-glucopyranoside (abbreviated DF3G). Seven metabolites in rat feces, urine and bile were firstly identified on the basis of their MS fragmentation behaviors. Three metabolites were identified in the feces, 6 in the urine and 2 in the bile, which suggested that demethylation, deglycosylation and deglycosylation followed by glucuronide conjugation were the major metabolic pathways for DF3G in vivo. Hydrolyzation might be the first step in the absorption and metabolism of DF3G. The possible metabolic pathway was proposed for the first time. The established method was simple, reliable and sensitive, revealing that it could be used to rapidly screen and identify the structures of metabolites of DF3G to better understand its metabolism in vivo.

Targeted analysis of conjugated and microbial-derived phenolic metabolites in human urine after consumption of an almond skin phenolic extract.

A single-blind, placebo-controlled, and randomized trial study was carried out with 16 healthy volunteers (7 men and 5 women). The test group ingested an encapsulated almond skin phenolic extract (884 mg of total polyphenols/dose) containing flavan-3-ols, flavonols, and flavanones, whereas the placebo group ingested microcrystalline cellulose. Our aim in this study was to determine changes in the urinary excretion of conjugated and microbial-derived phenolic metabolites before (-2 to 0 h) and after (0-2, 2-6, 6-10, and 10-24 h) intake of the almond polyphenols compared with the placebo group. For the test group, maximum urinary excretion of (epi)catechin and naringenin conjugates derived from phase II metabolism was attained at 2-6 h after consumption of the almond skin extract and excretions differed from the placebo group during this time period (P ≤ 0.0001). However, excretion of conjugated metabolites of isorhamnetin was highest at 10-24 h and did not differ from the placebo group during this time (P > 0.05). Hydroxyphenylvalerolactones reached maximum urinary levels at 6-10 h after consumption of almond polyphenols, and excretion differed from the placebo group during this time period (P = 0.0004). For the test group, excretions of phenolic acids (hydroxyphenylpropionic, hydroxyphenylacetic, hydroxybenzoic, and hydroxycinnamic acids) did not differ from the placebo group at any time period of urine collection (P > 0.05). The findings presented in this work provide evidence concerning the bioavailability of almond skin polyphenols considering the effects of both phase II and microbial metabolism.

Flavonol glycosides of sea buckthorn (Hippophaë rhamnoides ssp. sinensis) and lingonberry (Vaccinium vitis-idaea) are bioavailable in humans and monoglucuronidated for excretion.

Glucuronidation and excretion of sea buckthorn and lingonberry flavonols were investigated in a postprandial trial by analyzing the intact forms of flavonol glycosides as well as glucuronides in plasma, urine, and feces. Four study subjects consumed sea buckthorn (study day 1) and lingonberry (study day 2) breakfasts, and blood, urine, and fecal samples were collected for 8, 24, and 48 h, respectively. Both glycosides and glucuronides of the flavonol quercetin as well as kaempferol glucuronides were detected in urine and plasma samples after the consumption of lingonberries; 14% of flavonols in urine were glycosides, and 86% were glucuronidated forms (wt %). After the consumption of sea buckthorn, 5% of flavonols excreted in urine were detected intact, and 95% as the glucuronides (wt %). Solely glucuronides of flavonols isorhamnetin and quercetin were found in plasma after the consumption of sea buckthorn berries. Only glycosides were detected in the feces after each berry trial. Flavonol glycosides and glucuronides remained in blood and urine quite long, and the peak concentrations appeared slightly later than previously described. The berries seemed to serve as a good flavonol supply, providing steady flavonol input for the body for a relatively long time.

Absorption, metabolism, and excretion of green tea flavan-3-ols in humans with an ileostomy.

Green tea containing 634 micromol of flavan-3-ols was ingested by human subjects with an ileostomy. Ileal fluid, plasma, and urine collected 0-24 h after ingestion were analysed by HPLC-MS. The ileal fluid contained 70% of the ingested flavan-3-ols in the form of parent compounds (33%) and 23 metabolites (37%). The main metabolites effluxed back into the lumen of the small intestine were O-linked sulphates and methyl-sulphates of (epi)catechin and (epi)gallocatechin. Thus, in subjects with a functioning colon substantial quantities of flavan-3-ols would pass from the small to the large intestine. Plasma contained 16 metabolites, principally methylated, sulphated, and glucuronidated conjugates of (epi)catechin and (epi)gallocatechin, exhibiting 101-256 nM peak plasma concentration and the time to reach peak plasma concentration ranging from 0.8 to 2.2 h. Plasma pharmacokinetic profiles were similar to those obtained with healthy subjects, indicating that flavan-3-ol absorption occurs in the small intestine. Ileostomists had earlier plasma time to reach peak plasma concentration values than subjects with an intact colon, indicating the absence of an ileal brake. Urine contained 18 metabolites of (epi)catechin and (epi)gallocatechin in amounts corresponding to 6.8+/-0.6% of total flavan-3-ol intake. However, excretion of (epi)catechin metabolites was equivalent to 27% of the ingested (-)-epicatechin and (+)-catechin.

SPE-HPLC method for the determination of four flavonols in rat plasma and urine after oral administration of Abelmoschus manihot extract.

A SPE-HPLC method was developed and validated for the simultaneous determination of flavonols, isoquercitrin (1), hibifolin (2), myricetin (3), quercetin-3'-O-d-glucoside (4) and quercetin (5) in rat plasma and urine after oral administration of the total flavonoids from Abelmoschus manihot (TFA). The astragalin (6) and kaempferol (7) were used as internal standards (IS). Plasma and urine samples were pretreated by solid-phase extraction using Winchem C(18) reversed-phase cartridges. Analysis of the plasma and urinary extract was performed on YMC-Pack ODS-A C(18) and Thermo ODS-2HYEPRSIL C(18) reversed-phase column, respectively and a mobile phase of acetonitrile-0.1% phosphoric acid was employed. HPLC analysis was conducted with different elution gradients. The flow rate was 1.0 mL/min and the detection wavelength was set at 370 nm. Calibration ranges in plasma for flavonols 2-5 were at 0.011-2.220, 0.014-2.856, 0.022-4.320, and 0.028-5.600 microg/mL, respectively. In urine calibration ranges for flavonols 1, 2, 4 and 5 were at 2.00-16.00, 8.56-102.72, 2.70-21.60, and 3.00-24.00 microg/mL, respectively. The RSD of intra- and inter-day was less than 5.40% and 4.89% in plasma, and less than 3.96% and 6.85% in urine for all the analyses. A preliminary experiment to investigate the plasma concentration and urinary excretion of the flavonols after oral administration of TFA to rats demonstrated that the present method was suitable for determining the flavonols in rat plasma and urine.

Bioavailability and antioxidant activity of tea flavanols after consumption of green tea, black tea, or a green tea extract supplement.

BACKGROUND: Green and black tea polyphenols have been extensively studied as cancer chemopreventive agents. Many in vitro experiments have supported their strong antioxidant activity. Additional in vivo studies are needed to examine the pharmacokinetic relation of absorption and antioxidant activity of tea polyphenols administered in the form of green or black tea or tea extract supplements. OBJECTIVE: The purpose of this study was to compare the pharmacokinetic disposition of tea polyphenols and their effect on the antioxidant capacity in plasma 8 h after a bolus consumption of either green tea, black tea, or a green tea extract supplement. DESIGN: Thirty healthy subjects were randomly assigned to 3 different sequences of green tea, black tea, or a green tea extract supplement in a 3 x 3 crossover design with a 1-wk washout period in between treatments. RESULTS: Flavanol absorption was enhanced when tea polyphenols were administered as a green tea supplement in capsule form and led to a small but significant increase in plasma antioxidant activity compared with when tea polyphenols were consumed as black tea or green tea. All 3 interventions provided similar amounts of (-)-epigallocatechin-3-gallate. CONCLUSIONS: Our observations suggest that green tea extract supplements retain the beneficial effects of green and black tea and may be used in future chemoprevention studies to provide a large dose of tea polyphenols without the side effects of caffeine associated with green and black tea beverages.

Metabolic profiling of flavonol metabolites in human urine by liquid chromatography and tandem mass spectrometry.

Twenty-one flavonol metabolites have been identified by LC/ESI-MS/MS in human urine, including isomers, after the consumption of cooked onions. Metabolites identified include quercetin monoglucuronides, methyl quercetin monoglucuronides, a quercetin monoglucuronide sulfate, quercetin diglucuronides, a methyl quercetin diglucuronide, quercetin glucoside sulfates, methyl quercetin, quercetin, and kaempferol monoglucuronides. The fragmentation patterns of flavonol metabolites obtained by MS/MS were distinctive for some isomers, indicating that fragmentation patterns may be useful predictors of conjugation position. Two isomers of sulfate quercetin glucosides were also found in urine, suggesting that many of the quercetin glucosides in onion are absorbed intact and undergo metabolism to the sulfate conjugate. Additionally, the interindividual variation in urinary quercetin metabolite profiles was determined by comparing the relative level of six different quercetin metabolites excreted in the urine of healthy volunteers. The ranges of quercetin metabolites excreted were similar among volunteers, yet notable differences in the levels of metabolites among individuals were observed. This study demonstrates the potential of monitoring the range of quercetin metabolites to reveal information on interindividual biotransformation capacity in response to dietary manipulations and as a biomarker for flavonol consumption.