Food intake biomarkers for berries and grapes.

Grapes and berries are two types of widely consumed fruits characterized by a high content in different phytochemicals. However, their accurate dietary assessment is particularly arduous, because of the already wide recognized bias associated with self-reporting methods, combined with the large range of species and cultivars and the fact that these fruits are popularly consumed not only in fresh and frozen forms but also as processed and derived products, including dried and canned fruits, beverages, jams, and jellies. Reporting precise type and/or quantity of grape and berries in FFQ or diaries can obviously be affected by errors. Recently, biomarkers of food intake (BFIs) rose as a promising tool to provide accurate information indicating consumption of certain food items. Protocols for performing systematic reviews in this field, as well as for assessing the validity of candidate BFIs have been developed within the Food Biomarker Alliance (FoodBAll) Project. This paper aims to evaluate the putative BIFs for blueberries, strawberries, raspberries, blackberries, cranberries, blackcurrant, and grapes. Candidate BFIs for grapes were resveratrol metabolites and tartaric acid. The metabolites considered as putative BFI for berries consumption were mostly anthocyanins derivatives together with several metabolites of ellagitannins and some aroma compounds. However, identification of BFIs for single berry types encountered more difficulties. In the absence of highly specific metabolites reported to date, we suggested some multi-metabolite panels that may be further investigated as putative biomarkers for some berry fruits.

Biomarkers of intake for tropical fruits.

Consumption of fruit and vegetable is a key component of a healthy and sustainable diet. However, their accurate dietary assessment remains a challenge. Due to errors in self-reporting methods, the available dietary information is usually biased. Biomarkers of intake constitute objective tools to better reflect the usual or recent consumption of different foods, including fruits and vegetables. Partners of The Food Biomarker Alliance (FoodBall) Project have undertaken the task of reviewing the available literature on putative biomarkers of tropical fruit intake. The identified candidate biomarkers were subject to validation evaluation using eight biological and chemical criteria. This publication presents the current knowledge on intake biomarkers for 17 tropical fruits including banana, mango, and avocado as the most widely consumed ones. Candidate biomarkers were found only for banana, avocado, and watermelon. An array of banana-derived metabolites has been reported in human biofluids, among which 5-hydroxyindole-acetic acid, dopamine sulfate, methoxyeugenol glucuronide, salsolinol sulfate, 6-hydroxy-1-methyl-1,2,3,4-tetrahydro-ß-carboline-sulfate, and other catecholamine metabolites. Their validation is still at an early stage, with insufficient data on dose-response relationship. Perseitol and mannoheptulose have recently been reported as candidate biomarkers for avocado intake, while the amino acid citrulline has been associated with watermelon intake. Additionally, the examination of food composition data revealed some highly specific phytochemicals, which metabolites after absorption may be further studied as putative BFI for one or several tropical fruits. To make the field move forward, untargeted metabolomics, as a data-driven explorative approach, will have to be applied in both intervention and observational studies to discover putative BFIs, while their full validation and the establishment of dose-response calibration curves will require quantification methods at a later stage.

Validation of biomarkers of food intake-critical assessment of candidate biomarkers.

Biomarkers of food intake (BFIs) are a promising tool for limiting misclassification in nutrition research where more subjective dietary assessment instruments are used. They may also be used to assess compliance to dietary guidelines or to a dietary intervention. Biomarkers therefore hold promise for direct and objective measurement of food intake. However, the number of comprehensively validated biomarkers of food intake is limited to just a few. Many new candidate biomarkers emerge from metabolic profiling studies and from advances in food chemistry. Furthermore, candidate food intake biomarkers may also be identified based on extensive literature reviews such as described in the guidelines for Biomarker of Food Intake Reviews (BFIRev). To systematically and critically assess the validity of candidate biomarkers of food intake, it is necessary to outline and streamline an optimal and reproducible validation process. A consensus-based procedure was used to provide and evaluate a set of the most important criteria for systematic validation of BFIs. As a result, a validation procedure was developed including eight criteria, plausibility, dose-response, time-response, robustness, reliability, stability, analytical performance, and inter-laboratory reproducibility. The validation has a dual purpose: (1) to estimate the current level of validation of candidate biomarkers of food intake based on an objective and systematic approach and (2) to pinpoint which additional studies are needed to provide full validation of each candidate biomarker of food intake. This position paper on biomarker of food intake validation outlines the second step of the BFIRev procedure but may also be used as such for validation of new candidate biomarkers identified, e.g., in food metabolomic studies.

One-month exposure to soy isoflavones did not induce the ability to produce equol in postmenopausal women.

OBJECTIVE: As more and more postmenopausal women are taking soy isoflavone supplementation for relieving menopausal symptoms, we investigated the impact of chronic exposure on their bioavailability, with focus on achievable plasma concentrations and potential stimulation of the capacity to produce equol. SUBJECTS: A total of 12 Caucasian postmenopausal women. INTERVENTION: Volunteers ingested 100 mg isoflavones/day (aglycone equivalents, in cereal bars and yoghurts) for 1 month. Plasma concentrations of metabolites at 2, 4, 6, 8, 10, 12 and 24 h postdose, as well as urinary excretion in fractions over 36 h were compared between days 1 and 30. RESULTS: Similar plasma kinetic curves were obtained at day 1 and day 30 for genistein and daidzein. Maximum plasma concentrations were 1.68+/-0.68 micromol/l on day 1 compared to 2.27+/-0.76 micromol/l on day 30 for daidzein (P=0.056), and 3.88+/-1.50 micromol/l on day 1 compared to 5.30+/-2.38 micromol/l on day 30 for genistein (P=0.091). Urinary excretion of daidzein and genistein did not differ significantly between days 1 and 30. Maximum plasma concentration of equol increased significantly from 0.31+/-0.27 to 0.99+/-0.51 micromol/l for equol-producer volunteers (P=0.046). However, the seven volunteers who were classified as non-equol producers on day 1 did not acquire the ability to produce equol after 1-month exposure. CONCLUSIONS: Chronic exposure to isoflavones in postmenopausal women resulted in plasma concentrations as high as 2.5-5 micromol/l of each isoflavone, but did not induce the ability to produce equol.

The bioavailability of polyphenols is highly governed by the capacity of the intestine and of the liver to secrete conjugated metabolites.

BACKGROUND: After ingestion of a complex meal containing foods and beverages of plant origin, different polyphenols are likely to be simultaneously present in the intestine. However, almost nothing is known about their interactions and possible consequences on their bioavailability. AIM OF THE STUDY: The present study deals with the intestinal absorption and splanchnic metabolism of three polyphenols, genistein, hesperetin and ferulic acid (FA),when perfused in the small intestine alone or in combination, at different doses (15 and 120 microM). METHODS: The fate of polyphenols in the small intestine was studied using a rat in situ intestinal perfusion model. Polyphenols were analysed in perfusate, bile and plasma by HPLC. RESULTS: Whatever the perfused dose, the efficiency of the net transfer towards the enterocyte was similar for the three polyphenols and not significantly modified by any association between these molecules. However, FA largely differed from the two flavonoids by its low intestinal secretion of conjugates. When perfused at 15 microM, the secretion of conjugates back to the lumen represented 6.2% of the net transfer into the enterocytes for FA compared to 25.5 and 20 % for genistein and hesperetin respectively. Intestinal conjugation and secretion of conjugates back to the gut lumen varied with the dose of flavonoids: saturation of conjugation was observed for the highest dose or when a high dose of a second flavonoid was perfused simultaneously. Intensity of the biliary secretion substantially differed among tested polyphenols: 7.7% of the net transfer for FA vs 50% for genistein or hesperetin. The extent of the enterohepatic cycling of these polyphenols was proportional to the perfused dose and unaffected by the simultaneous presence of different compounds in the intestine. CONCLUSION: Genistein and hesperetin appeared less available than FA for peripheral tissues because of a high intestinal and biliary secretion of their conjugates. Moreover, data suggest that a high polyphenol intake may improve their bioavailability due to saturation of the intestinal secretion of conjugates.

Bioavailability in humans of the flavanones hesperidin and narirutin after the ingestion of two doses of orange juice.

OBJECTIVE: Flavanones are polyphenols specific of citrus fruits, where they are present in high amounts. Although citrus fruits and juices are widely consumed in the world, little information has been published on flavanone bioavailability in humans. The aim of the present study was to determine the nature of the circulating metabolites, the plasma kinetics and the urinary excretion patterns of the flavanones, hesperidin and narirutin. DESIGN: After an overnight fast, five healthy volunteers ingested 0.5 or 1 l of a commercial orange juice providing 444 mg/l hesperidin and 96.4 mg/l narirutin, together with a polyphenol-free breakfast. Blood was sampled at 10 different timepoints over a 24 h period. Urine was collected for 48 h, in five fractions. RESULTS: Flavanones metabolites appeared in plasma 3 h after the juice ingestion, reached a peak between 5 and 7 h, then returned to baseline at 24 h. The peak plasma concentration of hesperetin was 0.46+/-0.07 micro mol/l and 1.28+/-0.13 micro mol/l after the 0.5 and 1 l intake, respectively. It was lower for naringenin: 0.20+/-0.04 micro mol/l after the 1 l dose. The circulating forms of hesperetin were glucuronides (87%) and sulphoglucuronides (13%). For both flavanones, the urinary excretion was nearly complete 24 h after the orange juice ingestion. The relative urinary excretion was similar for hesperetin and naringenin and did not depend on the dose: values ranged from 4.1+/-1.2 to 7.9+/-1.7% of the intake. CONCLUSIONS: In case of a moderate or high consumption of orange juice, flavanones may represent an important part of the pool of total polyphenols present in plasma.

Bioavailability of phloretin and phloridzin in rats.

Phloretin is a flavonoid found exclusively in apples and in apple-derived products where it is present as the glucosidic form, namely, phloridzin (phloretin 2'-O-glucose). In the present study, we compared the changes in plasma and urine concentrations of these two compounds in rats fed a single meal containing 0.25% phloridzin or 0.157% phloretin (corresponding to the ingestion of 22 mg of phloretin equivalents). In plasma, phloretin was recovered mainly as the conjugated forms (glucuronided and/or sulfated) but some unconjugated phloretin was also detected. By contrast, no trace of intact phloridzin was detected in plasma of rats fed a phloridzin meal. These compounds presented different kinetics of absorption; phloretin appeared more rapidly in plasma when rats were fed the aglycone than when fed the glucoside. However, whatever compound was administered, no significant difference in the plasma concentrations of total phloretin were observed 10 h after food intake. At 24 h after the beginning of the meal, the plasma concentrations of phloretin were almost back to the baseline, indicating that this compound was excreted rapidly in urine. The total urinary excretion rate of phloretin was not affected by the forms administered, and was estimated to be 8.5 micromol/24 h in rats fed phloretin or phloridzin. Thus, 10.4% of the ingested dose was recovered in urine after 24 h.

Comparison of the intestinal absorption of quercetin, phloretin and their glucosides in rats.

Absorption and metabolism of quercetin and isoquercitrin (quercetin 3-O-glucose) were investigated in rats after in situ perfusion of jejunum plus ileum (15 nmol/min) for 30 min and compared with those of phloretin and phloridzin (phloretin 2'-O-glucose). After perfusion of the glucosides, the corresponding aglycone forms and conjugated derivatives appeared in the lumen. The conjugated metabolites were similar to those recovered after intestinal perfusion of the aglycone forms. Regardless of the aglycone or glucoside perfused, only conjugated forms were present in the mesenteric vein blood draining the perfused segment showing the importance of intestinal conjugation. The hydrolysis of glucosides was a prerequisite step before their conjugation by intestinal enzymes and their transport towards the mucosal and serosal sides. In contrast to phloridzin, lactase phloridzin hydrolase activity did not seem to be an essential pathway for isoquercitrin hydrolysis. The 3-O-glucosylation of quercetin improved the net absorption of the aglycone (P < 0.05), whereas phloretin absorption decreased when present as 2'-O-glucoside (P < 0.05). Whatever the perfused compound, the efficiency of the absorption seemed to be linked to the intestinal conjugation process and to the luminal secretion of metabolites.

Catechin is metabolized by both the small intestine and liver of rats.

Flavan-3-ols are the most abundant flavonoids in the human diet, but little is known about their absorption and metabolism. In this study, the absorption and metabolism of the monomeric flavan-3-ol, catechin, was investigated after the in situ perfusion of the jejunum + ileum in rats. Five concentrations of catechin were studied, ranging from 1 to 100 micromol/L. The absorption of catechin was directly proportional to the concentration, and 35 +/- 2% of the perfused catechin was absorbed during the 30-min period. Effluent samples contained only native catechin, indicating that intestinal excretion of metabolites is not a mechanism of catechin elimination. Catechin was absorbed into intestinal cells and metabolized extensively because no native catechin could be detected in plasma from the mesenteric vein. Mesenteric plasma contained glucuronide conjugates of catechin and 3'-O-methyl catechin (3'OMC), indicating the intestinal origin of these conjugates. Additional methylation and sulfation occurred in the liver, and glucuronide + sulfate conjugates of 3'OMC were excreted extensively in bile. Circulating forms were mainly glucuronide conjugates of catechin and 3'OMC. The data further demonstrate the role of the rat small intestine in the glucuronidation and methylation of flavonoids as well as the role of the liver in sulfation, methylation and biliary excretion.

Bioavailability of the flavanone naringenin and its glycosides in rats.

Naringenin, the predominant flavanone in grapefruit, mainly occurs as glycosides such as naringenin-7- rhamnoglucoside or naringenin-7-glucoside. This study compared kinetics of absorption of naringenin and its glycosides in rats either after a single flavanone-containing meal or after adaptation to a diet for 14 days. Regardless of the diet, circulating metabolites were glucurono- and sulfoconjugated derivatives of naringenin. The kinetics of absorption of naringenin and naringenin-7-glucoside were similar, whereas naringenin-7-rhamnoglucoside exhibited a delay in its intestinal absorption, resulting in decreased bioavailability. After naringenin-7-glucoside feeding, no glucoside was found in the cecum. However, after feeding naringenin-7-rhamnoglucoside, some naringenin-7-rhamnoglucoside accumulated in cecum before being hydrolyzed by intestinal microflora. Adaptation to flavanone diets did not induce accumulation of plasma naringenin. Moreover, flavanone cecal content markedly decreased after adaptation, and almost no naringenin-7-rhamnoglucoside was recovered after naringenin-7-rhamnoglucoside feeding, suggesting that an adaptation of cecal microflora had occurred. Overall, these data indicate that flavanones are efficiently absorbed after feeding to rats and that their bioavailability is related to their glycosidic moiety.

Respective bioavailability of quercetin aglycone and its glycosides in a rat model.

A large number of flavonoids, mostly O-glycosides, are found in foods of plant origin. The bound sugar moiety is known to influence their bioavailability. We examined here the effect of the nature of the sugar on the absorption of the glycosides. Four groups of rats (n = 6) received a meal containing 20 mg of quercetin equivalents supplied as aglycone, quercetin 3-glucoside, quercetin 3-rhamnoside or rutin. Plasma were hydrolysed by a beta-glucuronidase/sulfatase and analyzed by HPLC coupled to UV detection at 370 nm. Four hours after the beginning of the meal, the quercetin metabolites present in plasma were identical in all groups but their total concentrations were quite different. With pure quercetin the circulating levels were 1.7 +/- 1.8 microM, but this level was three fold higher when quercetin was supplied as quercetin 3-glucoside (33.2 +/- 3.5 microM). By contrast, the plasma concentrations of quercetin metabolites was quite low with the rutin meal (about 3 microM) and undetectable after the quercetin 3-rhamnoside meal. These data suggest that the 3-O-glucosylation improves the absorption of quercetin in the small intestine, whereas the binding of a rhamnose or of a glucose-rhamnose moiety to the aglycone markedly depressed its absorption. Additionnal experiments have shown that the higher plasma levels measured after the meal containing quercetin 3-glucoside compared to quercetin were maintained throughout a 24 hour period following the meal. In conclusion, the nature of the glycosylation markedly influences the efficiency of quercetin absorption in rats. Quercetin 3-glucose can be absorbed in the small intestine and is better absorbed than quercetin itself. By contrast, glycosides containing a rhamnose moiety could not be absorbed in the small intestine.

Quercetin 3-O-beta-glucoside is better absorbed than other quercetin forms and is not present in rat plasma.

The effect of the nature of the sugar moiety on quercetin absorption has been investigated in rats. Four groups of rats received an experimental meal containing 20 mg of quercetin equivalents, supplied as quercetin, quercetin 3-O-beta-glucoside, quercetin 3-O-beta-rhamnoside or rutin. Four hours after the meal, the metabolites identified in hydrolysed plasma were identical in all groups (3'- and 4'-methylquercetin). However, the total concentration of metabolites was markedly different: 11.2+/-1.8, 2.5+/-2.0 and 33.2+/-3.5 microM for the quercetin, rutin, and quercetin 3-glucoside meals respectively. After quercetin 3-rhamnoside consumption, we failed to detect any metabolites in the plasma. These data suggest that the 3-O-glucosylation improves the absorption of quercetin in the small intestine, whereas the binding of a rhamnose to the aglycone markedly depresses it. Additional experiments have shown that the higher plasma levels measured after quercetin 3-glucoside meal compared to the quercetin meal were maintained throughout the 24-hour period following the meal. Using a multi-electrode coulometric detection, together with suitable chromatographic conditions, we were able to distinguish between the conjugated and the glycosylated forms. Thus, we clearly showed the absence of quercetin 3-O-beta-glucoside in the plasma from rats fed a diet containing this glucoside. This result suggests that quercetin 3-O-beta-glucoside is hydrolysed before or during its intestinal absorption.

Part of quercetin absorbed in the small intestine is conjugated and further secreted in the intestinal lumen.

Rutin and quercetin absorption and metabolism were investigated in rats after in situ perfusion of jejunum plus ileum (15 nmol/min). In contrast to rutin, a high proportion of quercetin (two-thirds) disappeared during perfusion, reflecting extensive transfer into the intestinal wall. Net quercetin absorption was not complete (2.1 nmol/min), inasmuch as 52% were reexcreted in the lumen as conjugated derivatives (7.7 nmol/min). Enterohepatic recycling contribution of flavonoids was excluded by catheterization of the biliary duct before perfusion. After a 30-min perfusion period, 0.71 microM of quercetin equivalents were detected in plasma, reflecting a significant absorption from the small intestine. The differential hydrolysis of effluent samples by glucuronidase and/or sulfatase indicates that the conjugated forms released in the lumen were 1) glucuronidated derivatives of quercetin and of its methoxylated forms (64%) and 2) sulfated form of quercetin (36%). In vitro quercetin glucuronides synthetized using jejunal and ileal microsomal fractions were similar to those recovered in the effluent of perfusion. These data suggest that glucuronidation and sulfatation take place in intestinal cells, whereas no glucurono-sulfoconjugates could be detected in the effluent. The present work shows that a rapid quercetin absorption in the small intestine is very effective together with its active conjugation in intestinal cells.

Comparison of the bioavailability of quercetin and catechin in rats.

Quercetin and catechin are present in noticeable amounts in human diet and these polyphenolic compounds are supposed to exert beneficial effects on human health. However, their metabolic fates in the organism have never been compared. In the present study, rats were fed a 0.25% quercetin or a 0.25% catechin diet. Quercetin and catechin metabolites were analyzed in plasma and liver samples by high-performance liquid chromatography coupled to an ultraviolet or a multielectrode coulometric detection. All plasma metabolites were present as conjugated forms, but catechin metabolites were mainly constituted by glucuronidated derivatives, whereas quercetin metabolites were sulfo- and glucurono-sulfo conjugates. Quercetin was more intensively methylated than catechin in plasma. The plasma quercetin metabolites are well maintained during the postabsorptive period (approximately 50 microM), whereas the concentration of catechin metabolites dropped dramatically between 12- and 24-h after an experimental meal (from 38.0 to 4.5 microM). In the liver, the concentrations of quercetin and catechin derivatives were lower than in plasma, and no accumulation was observed when the rats were adapted for 14 d to the supplemented diets. The hepatic metabolites were intensively methylated (90-95%), but in contrast to plasma, some free aglycones could be detected. Thus, it clearly appears that studies dealing with the biological impact of these polyphenols should take into account the feature of their bioavailability, particularly the fact that their circulating metabolites are conjugated derivatives.

Plasma metabolites of quercetin and their antioxidant properties.

Quercetin is one of the most widely distributed flavonoids present in fruits and vegetables. The present experiments were performed on rats adapted for 3 wk to a semipurified diet supplemented with 0.2% quercetin. The major part of the circulating metabolites of quercetin (91.5%) are glucurono-sulfo conjugates of isorhamnetin (3'-O-methyl quercetin; 89.1 +/- 2.1 microM) and of quercetin (14.7 +/- 1.7 microM); the minor part (8.5%) is constituted by glucuronides of quercetin and its methoxylated forms (9.6 +/- 2.3 microM). Conjugated dienes formation, resulting from Cu2+-catalyzed oxidation of rat very low density lipoproteins + low density lipoproteins (LDL), was effectively inhibited in vitro by conjugated metabolites of quercetin. These metabolites appeared to be four times more potent than trolox in inhibiting LDL oxidation. Moreover, the plasma from rats adapted to a diet containing 0.2% quercetin exhibited a total antioxidant status markedly higher than that of control rats (+60%). This study shows that ubiquitous quercetin is conjugated in vivo, yielding metabolites that exhibit antioxidant properties. Thus the health benefits of flavonoids in foods can be due to the antioxidant properties of their metabolites.

Quercetin is recovered in human plasma as conjugated derivatives which retain antioxidant properties.

Quercetin is one of the most abundant flavonoids in the human diet. This study aimed to determine the plasma concentrations of quercetin in 10 healthy volunteers after the consumption of a complex meal rich in plant products. Quercetin was determined in plasma (2 h before, and 3, 7 and 20 h after the meal), and in a duplicated portion of the meal by HPLC analysis with an electrochemical detection. The amount of ingested quercetin was estimated to be 87 mg. Before the meal, quercetin concentration in hydrolyzed plasmas ranged from 28 to 142 nM. A marked increase was observed 3 h after the meal in all subjects, with a mean concentration of 373 nM (S.E.M. = 61). After 7 h, quercetin concentration in hydrolyzed plasmas decreased and after 20 h basal levels were found again. The antioxidant capacities of quercetin, 3'-O-methylquercetin, and of some of their conjugated derivatives were compared by the measurement of the conjugated dienes resulting from the Cu2+-induced oxidation of human LDL. 3'-O-Methylquercetin and conjugated derivatives of quercetin significantly prolonged the lag phase, but the magnitude of their effect was about half that of the aglycone.

Bioavailability of rutin and quercetin in rats.

Quercetin is a powerful antioxidant which is widely distributed in edible plants, mainly as glycosides such as rutin. It has been reported to be absorbed in mammals, but its metabolism needs further investigation to evaluate its possible physiological effects. We compared the evolution of the absorption of quercetin and rutin in rats fed with supplemented diets. Rutin was absorbed more slowly than quercetin because it must be hydrolysed by the cecal microflora, whereas quercetin was absorbed from the small intestine. Conjugated derivatives of quercetin, and its methylated forms isorhamnetin and tamarixetin, were recovered in plasma from rats receiving the two kinds of experimental diets after the first meal, but after 10 days, no traces of tamarixetin were detected anymore. The rate of elimination of quercetin metabolites seems very low, and high plasma concentrations are easily maintained with a regular supply of quercetin or rutin in the diet.

Quercetin metabolites in plasma of rats fed diets containing rutin or quercetin.

We studied the bioavailability and the plasma transport of flavonols in rats fed quercetin or rutin diets. Wistar rats were fed one of the following purified diets for 10 d: control; 16.4 or 8.2 mmol rutin/kg diet; or 16.4, 8.2 or 4.1 mmol quercetin/kg diet. Flavonol concentrations were determined in plasma, ileal and cecal contents, and feces. In rats fed diets containing 16.4 mmol quercetin or rutin/kg, the concentration of circulating flavonols was approximately 115 mumol/L. Quercetin or rutin administration resulted in similar concentrations of quercetin in cecal contents. By HPLC analysis and beta-glucuronidase/sulfatase treatment, plasma flavonols have been identified as conjugated quercetin itself, or a conjugated form (4.5-fold as abundant) of an aglycone less polar than quercetin. Rats fed quercetin or rutin diets had a green/yellow-colored plasma that exhibited a peak absorbance at 411 nm, vs. 363 or 375 nm for pure rutin or quercetin solutions, respectively. This shift of band I absorption was obtained when pure quercetin was in the presence of albumin or added to a plasma fraction. The bathochromic properties of flavonoids in the presence of albumin are highly dependent on the presence of the C-2/C-3 double bond on the C-ring and are influenced by the degree of B-ring hydroxylation. The existence of intermolecular bonds between albumin and quercetin is supported by in vitro absorbance and fluorescence studies. With human albumin, the fluorescence intensity and the shift of quercetin absorbance increased in parallel to the albumin/quercetin molar ratio. Conjugated diene formation, resulting from Cu(2+)-catalyzed oxidation of human LDL or rat VLDL+LDL was effectively inhibited in vitro by 0.5 mumol/L quercetin. These results show that dietary flavonols are recovered in rat plasma as conjugated metabolites in non-negligible concentrations, and that these flavonols may be interesting antioxidant micronutrients with a variety of biological effects.