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.

The impact of ambient air pollution on the human blood metabolome.

BACKGROUND: Biological perturbations caused by air pollution might be reflected in the compounds present in blood originating from air pollutants and endogenous metabolites influenced by air pollution (defined here as part of the blood metabolome). We aimed to assess the perturbation of the blood metabolome in response to short term exposure to air pollution. METHODS: We exposed 31 healthy volunteers to ambient air pollution for 5h. We measured exposure to particulate matter, particle number concentrations, absorbance, elemental/organic carbon, trace metals, secondary inorganic components, endotoxin content, gaseous pollutants, and particulate matter oxidative potential. We collected blood from the participants 2h before and 2 and 18h after exposure. We employed untargeted metabolite profiling to monitor 3873 metabolic features in 493 blood samples from these volunteers. We assessed lung function using spirometry and six acute phase proteins in peripheral blood. We assessed the association of the metabolic features with the measured air pollutants and with health markers that we previously observed to be associated with air pollution in this study. RESULTS: We observed 89 robust associations between air pollutants and metabolic features two hours after exposure and 118 robust associations 18h after exposure. Some of the metabolic features that were associated with air pollutants were also associated with acute health effects, especially changes in forced expiratory volume in 1s. We successfully identified tyrosine, guanosine, and hypoxanthine among the associated features. Bioinformatics approach Mummichog predicted enriched pathway activity in eight pathways, among which tyrosine metabolism. CONCLUSIONS: This study demonstrates for the first time the application of untargeted metabolite profiling to assess the impact of air pollution on the blood metabolome.

The exposome in practice: Design of the EXPOsOMICS project.

EXPOsOMICS is a European Union funded project that aims to develop a novel approach to the assessment of exposure to high priority environmental pollutants, by characterizing the external and the internal components of the exposome. It focuses on air and water contaminants during critical periods of life. To this end, the project centres on 1) exposure assessment at the personal and population levels within existing European short and long-term population studies, exploiting available tools and methods which have been developed for personal exposure monitoring (PEM); and 2) multiple "omic" technologies for the analysis of biological samples (internal markers of external exposures). The search for the relationships between external exposures and global profiles of molecular features in the same individuals constitutes a novel advancement towards the development of "next generation exposure assessment" for environmental chemicals and their mixtures. The linkage with disease risks opens the way to what are defined here as 'exposome-wide association studies' (EWAS).

Plasma concentrations and intakes of amino acids in male meat-eaters, fish-eaters, vegetarians and vegans: a cross-sectional analysis in the EPIC-Oxford cohort.

BACKGROUND/OBJECTIVES: We aimed to investigate the differences in plasma concentrations and in intakes of amino acids between male meat-eaters, fish-eaters, vegetarians and vegans in the Oxford arm of the European Prospective Investigation into Cancer and Nutrition. SUBJECTS/METHODS: This cross-sectional analysis included 392 men, aged 30-49 years. Plasma amino acid concentrations were measured with a targeted metabolomic approach using mass spectrometry, and dietary intake was assessed using a food frequency questionnaire. Differences between diet groups in mean plasma concentrations and intakes of amino acids were examined using analysis of variance, controlling for potential confounding factors and multiple testing. RESULTS: In plasma, concentrations of 6 out of 21 amino acids varied significantly by diet group, with differences of -13% to +16% between meat-eaters and vegans. Concentrations of methionine, tryptophan and tyrosine were highest in fish-eaters and vegetarians, followed by meat-eaters, and lowest in vegans. A broadly similar pattern was seen for lysine, whereas alanine concentration was highest in fish-eaters and lowest in meat-eaters. For glycine, vegans had the highest concentration and meat-eaters the lowest. Intakes of all 18 dietary amino acids differed by diet group; for the majority of these, intake was highest in meat-eaters followed by fish-eaters, then vegetarians and lowest in vegans (up to 47% lower than in meat-eaters). CONCLUSIONS: Men belonging to different habitual diet groups have significantly different plasma concentrations of lysine, methionine, tryptophan, alanine, glycine and tyrosine. However, the differences in plasma concentrations were less marked than and did not necessarily mirror those seen for amino acid intakes.

Flavonoid and lignan intake in relation to bladder cancer risk in the European Prospective Investigation into Cancer and Nutrition (EPIC) study.

BACKGROUND: There is growing evidence of the protective role of dietary intake of flavonoids and lignans on cancer, but the association with bladder cancer has not been thoroughly investigated in epidemiological studies. We evaluated the association between dietary intakes of total and subclasses of flavonoids and lignans and risk of bladder cancer and its main morphological type, urothelial cell carcinoma (UCC), within the European Prospective Investigation into Cancer and Nutrition (EPIC) study. METHODS: A cohort of 477 312 men and women mostly aged 35-70 years, were recruited in 10 European countries. At baseline, dietary flavonoid and lignan intakes were estimated using centre-specific validated questionnaires and a food composition database based on the Phenol-Explorer, the UK Food Standards Agency and the US Department of Agriculture databases. RESULTS: During an average of 11 years of follow-up, 1575 new cases of primary bladder cancer were identified, of which 1425 were UCC (classified into aggressive (n=430) and non-aggressive (n=413) UCC). No association was found between total flavonoid intake and bladder cancer risk. Among flavonoid subclasses, significant inverse associations with bladder cancer risk were found for intakes of flavonol (hazard ratio comparing fifth with first quintile (HRQ5-Q1) 0.74, 95% confidence interval (CI): 0.61-0.91; P-trend=0.009) and lignans (HRQ5-Q1 0.78, 95% CI: 0.62-0.96; P-trend=0.046). Similar results were observed for overall UCC and aggressive UCC, but not for non-aggressive UCC. CONCLUSIONS: Our study suggests an inverse association between the dietary intakes of flavonols and lignans and risk of bladder cancer, particularly aggressive UCC.

Physical activity, sex steroid, and growth factor concentrations in pre- and post-menopausal women: a cross-sectional study within the EPIC cohort.

PURPOSE: Increased physical activity (PA) is associated with a reduced risk of several cancers. PA may reduce cancer risk by changing endogenous hormones levels, but relatively little research has focused on this topic. The purpose of this study was to elucidate the relation between PA and endogenous hormone concentrations. METHODS: A cross-sectional analysis of 798 pre- and 1,360 post-menopausal women included as controls in case-control studies on endogenous hormones (steroids, progesterone, sex-hormone-binding globulin (SHBG), and growth factors) levels, and cancer risk nested within European Prospective Investigation into Cancer and Nutrition cohort was performed. Multivariate regression analyses were performed to compare geometric mean levels of hormones and SHBG by categories of PA. RESULTS: In pre-menopausal women, active women had 19 % significantly lower concentrations of androstenedione, 14 % lower testosterone, and 20 % lower free testosterone than inactive women, while no differences were observed for estrogens, progesterone, SHBG, and growth factors. In post-menopausal women, active women had 18 % significantly lower estradiol and 20 % lower free estradiol concentrations than inactive women, while no differences were observed for the other hormones and SHBG. More vigorous forms of physical activity were associated with higher insulin-like growth factor-I concentrations. Adjustment for body mass index did not alter the associations. Overall, the percentage of variance in hormone concentrations explained by PA levels was <2 %. CONCLUSIONS: Our results support the hypothesis of an influence, although small in magnitude, of PA on sex hormone levels in blood, independent of body size.

Impact of thearubigins on the estimation of total dietary flavonoids in the European Prospective Investigation into Cancer and Nutrition (EPIC) study.

Thearubigins (TR) are polymeric flavanol-derived compounds formed during the fermentation of tea leaves. Comprising ∼70% of total polyphenols in black tea, TR may contribute majorly to its beneficial effects on health. To date, there is no appropriate food composition data on TR, although several studies have used data from the US Department of Agriculture (USDA) database to estimate TR intakes. We aimed to estimate dietary TR in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort and assess the impact of including TR or not in the calculation of the total dietary flavonoid intake. Dietary data were collected using a single standardized 24-h dietary recall interviewer-administered to 36 037 subjects aged 35-74 years. TR intakes were calculated using the USDA database. TR intakes ranged from 0.9 mg/day in men from Navarra and San Sebastian in Spain to 532.5 mg/day in men from UK general population. TR contributed <5% to the total flavonoid intake in Greece, Spain and Italy, whereas in the UK general population, TR comprised 48% of the total flavonoids. High heterogeneity in TR intake across the EPIC countries was observed. This study shows that total flavonoid intake may be greatly influenced by TR, particularly in high black tea-consuming countries. Further research on identification and quantification of TR is needed to get more accurate dietary TR estimations.

Bilberry anthocyanin-rich extract alters expression of genes related to atherosclerosis development in aorta of apo E-deficient mice.

Intake of anthocyanin-rich foods has been associated with a reduced risk of cardiovascular diseases. We recently reported that a nutritional supplementation with a bilberry anthocyanin-rich extract (BE) attenuates atherosclerotic lesion development in apolipoprotein E-deficient (apoE⁻/⁻) mice. However, the mechanism(s) of their preventive action are not completely understood. Anthocyanins may alter mRNA levels of genes related to atherosclerosis in cultured macrophages and endothelial cells, but in vivo studies remain scarce. The aim of the present study was to explore the in vivo mechanisms of action of the same bilberry extract, administered by supplementation at a nutritional level, in the aorta of apo E⁻/⁻ mice using a global transcriptomic approach. This study focused on the early stage of atherosclerosis development for better assessment of BE action on initiation mechanisms of this pathology. After a two week period, plasma lipid and antioxidant capacity were evaluated and the global genomic analysis was carried out using pangenomic microarrays. BE supplementation significantly improved hypercholesterolemia whereas the plasmatic antioxidant status remained unchanged. Nutrigenomic analysis identified 1261 genes which expression was modulated by BE in the aorta. Bioinformatic analysis revealed that these genes are implicated in different cellular processes such as oxidative stress, inflammation, transendothelial migration and angiogenesis, processes associated with atherosclerosis development/protection. Some of the most significantly down-regulated genes included genes coding for AOX1, CYP2E1 or TXNIP implicated in the regulation of oxidative stress, JAM-A coding for adhesion molecules or VEGFR2 implicate in regulation of angiogenesis. Other genes were up-regulated, such as CRB3, CLDN14 or CDH4 potentially associated with increased cell-cell adhesion and decreased paracellular permeability. These results provide a global integrated view of the mechanisms involved in the preventive action of bilberry anthocyanin-rich extract against atherosclerosis.

Identification of the 100 richest dietary sources of polyphenols: an application of the Phenol-Explorer database.

BACKGROUND/OBJECTIVES: The diversity of the chemical structures of dietary polyphenols makes it difficult to estimate their total content in foods, and also to understand the role of polyphenols in health and the prevention of diseases. Global redox colorimetric assays have commonly been used to estimate the total polyphenol content in foods. However, these assays lack specificity. Contents of individual polyphenols have been determined by chromatography. These data, scattered in several hundred publications, have been compiled in the Phenol-Explorer database. The aim of this paper is to identify the 100 richest dietary sources of polyphenols using this database. SUBJECTS/METHODS: Advanced queries in the Phenol-Explorer database (www.phenol-explorer.eu) allowed retrieval of information on the content of 502 polyphenol glycosides, esters and aglycones in 452 foods. Total polyphenol content was calculated as the sum of the contents of all individual polyphenols. These content values were compared with the content of antioxidants estimated using the Folin assay method in the same foods. These values were also extracted from the same database. Amounts per serving were calculated using common serving sizes. RESULTS: A list of the 100 richest dietary sources of polyphenols was produced, with contents varying from 15,000 mg per 100 g in cloves to 10 mg per 100 ml in rosé wine. The richest sources were various spices and dried herbs, cocoa products, some darkly coloured berries, some seeds (flaxseed) and nuts (chestnut, hazelnut) and some vegetables, including olive and globe artichoke heads. A list of the 89 foods and beverages providing more than 1 mg of total polyphenols per serving was established. A comparison of total polyphenol contents with antioxidant contents, as determined by the Folin assay, also showed that Folin values systematically exceed the total polyphenol content values. CONCLUSIONS: The comprehensive Phenol-Explorer data were used for the first time to identify the richest dietary sources of polyphenols and the foods contributing most significantly to polyphenol intake as inferred from their content per serving.

The regular consumption of a polyphenol-rich apple does not influence endothelial function: a randomised double-blind trial in hypercholesterolemic adults.

BACKGROUND/OBJECTIVES: Epidemiological studies suggest that apple consumption is associated with a reduction in cardiovascular disease risk. Apple polyphenols may contribute to explain these effects. Endothelial dysfunction has been associated with early stage of atherosclerosis and polyphenols from various dietary sources have been shown to reverse it. The aim of the present study was to investigate the effect of the consumption of a polyphenol-rich apple on endothelial function. SUBJECTS/METHODS: In all, 30 hypercholesterolemic volunteers were included in a double-blind, randomized crossover trial. They successively consumed 40 g of two lyophilized apples, polyphenol-rich and polyphenol-poor, providing respectively 1.43 and 0.21 g polyphenols per day during two 4-week periods separated by a 4-week washout period. RESULTS: Brachial artery flow-mediated vasodilation (FMD) was assessed at the beginning and at the end of each intervention period. FMD did not differ between the polyphenol-rich and the polyphenol-poor apples, neither did the other cardiovascular disease risk factors (plasma lipids, homocysteine, antioxidant capacity). CONCLUSIONS: These data suggest that over a 4-week period, the consumption of a polyphenol-rich apple does not improve vascular function in hypercholesterolemic patients.

Phenol-Explorer: an online comprehensive database on polyphenol contents in foods.

A number of databases on the plant metabolome describe the chemistry and biosynthesis of plant chemicals. However, no such database is specifically focused on foods and more precisely on polyphenols, one of the major classes of phytochemicals. As antioxidants, polyphenols influence human health and may play a role in the prevention of a number of chronic diseases such as cardiovascular diseases, some cancers or type 2 diabetes. To determine polyphenol intake in populations and study their association with health, it is essential to have detailed information on their content in foods. However this information is not easily collected due to the variety of their chemical structures and the variability of their content in a given food. Phenol-Explorer is the first comprehensive web-based database on polyphenol content in foods. It contains more than 37,000 original data points collected from 638 scientific articles published in peer-reviewed journals. The quality of these data has been evaluated before they were aggregated to produce final representative mean content values for 502 polyphenols in 452 foods. The web interface allows making various queries on the aggregated data to identify foods containing a given polyphenol or polyphenols present in a given food. For each mean content value, it is possible to trace all original content values and their literature sources. Phenol-Explorer is a major step forward in the development of databases on food constituents and the food metabolome. It should help researchers to better understand the role of phytochemicals in the technical and nutritional quality of food, and food manufacturers to develop tailor-made healthy foods. Database URL: http://www.phenol-explorer.eu.

Contribution of genomics to the understanding of physiological functions.

Genomics has brought with it a true biological revolution and can be applied to all areas of life sciences. The advent of genomics is thus linked to the development of high-throughput techniques which allows the genome of organisms as a whole to be studied. The first high-throughput techniques to be developed were sequencing methods. These advances will allow new approaches to a variety of problems in biology. For instance, the emerging fields of genomic medicine in humans and genomic selection in livestock are promising. After the sequencing of genomes, genomics has shifted to the study of gene expression and function. This is called the "post-genomic area" by some authors or "functional genomics" by others. The most recent "omics" to be developed are associated with the study of the metabolism (e.g. metabolomics). Integrative "omics" approaches (e.g. nutrigenomics) are based on the association of the omics tools at different levels (DNA, RNA, proteins, metabolites) for a specific objective (here nutrition). In terms of perspectives, it is likely that methods for collecting data will outstrip our capacity to adequately analyse these data. So scientists must develop bioinformatic tools and methods to overcome this difficulty. In addition, high-throughput techniques need to be developed in physiology in order to match the increasing amount of genomic information with true biological data. Finally, there is no doubt that all these new approaches will allow important new genes and novel biological mechanisms to be discovered. Physiological models with invalidated or over-expressed genes will be precious tools to check these new biological discoveries.

Urinary excretion of 13 dietary flavonoids and phenolic acids in free-living healthy subjects - variability and possible use as biomarkers of polyphenol intake.

OBJECTIVE: Estimation of dietary intake of polyphenols is difficult, due to limited availability of food composition data and bias inherent to dietary assessment methods. The aim of the present study was to evaluate whether we could detect polyphenols and their metabolites in a spot urine sample in a free-living human population and whether it was related to those observed in 24-h urine samples, for potential use as a biomarkers of polyphenol intake. SUBJECTS: Four 24-h urine samples and two spot urine samples were collected from 154 participants of the SU.VI.MAX cohort (a randomized primary-prevention trial evaluating the effect of daily antioxidant supplementation on chronic diseases) in two separate studies over, respectively, a 7- and 2-day periods. Thirteen polyphenols and metabolites (chlorogenic acid (CGA), caffeic acid (CA), m-coumaric acid (mCOU), gallic acid (GA), 4-O-methylgallic acid (MeGA), quercetin (Q), isorhamnetin (MeQ), kaempferol (K), hesperetin (HESP), naringenin (NAR), phloretin (PHLOR), enterolactone (ENL) and enterodiol (END) were measured using HPLC-ESI-MS-MS. RESULTS: Correlations between the urinary excretion levels were observed. The most significant were explained by metabolic filiations (CGA/CA, CA/mCOU, GA/MeGA, Q/MeQ, NAR/PHLOR, ENL/END) or co-occurrence in a same food source (NAR/HESP). Concentrations in spot samples correlated with those in 24-h urine sample (P<0.02, except for CA and for MeQ). Intra-individual variations were smaller than inter-individual variations for all polyphenols (P<0.01) except for MeGA and for PHLOR. CONCLUSION: These results show that these polyphenols and metabolites are useful biomarkers for polyphenol intake.

Microbial metabolism of caffeic acid and its esters chlorogenic and caftaric acids by human faecal microbiota in vitro.

Caffeic acid and its esters, chlorogenic and caftaric acids, are major dietary polyphenols present in various foods and beverages. Although caffeic acid is easily absorbed in the small intestine, its esterification with quinic acid, as in chlorogenic acid, decreases its gut absorption and increases the quantities reaching the colon and its microbiota. The microbial conversion of caftaric acid, the tartaric acid ester of caffeic acid, has not been studied earlier. In this work we compared the direct action of a human faecal microbiota on the metabolism of caffeic, chlorogenic and caftaric acids in an in vitro fermentation model. All substrates disappeared quickly and none of the free acids (caffeic, quinic or tartaric acids) were detected after 2 hours of incubation. Two major microbial metabolites were identified by HPLC-ESI-MS-MS as 3-hydroxyphenylpropionic (3-HPP) and benzoic acids (BA). Maximal levels of 3-HPP were reached after 2 h of fermentation and accounted for 9-24% of the dose of caffeic acid and its esters. BA was formed steadily throughout the incubation, accounting for 4-5% of the initial dose of the substrates after 24 h of incubation. The similarities in the metabolic patterns observed for caffeic, chlorogenic and caftaric acids suggest that esterification does not influence the metabolism of caffeic acid by the gut microbiota.

Flavanone metabolism in healthy and tumor-bearing rats.

Flavanones, the main polyphenols of citrus fruits, are thought to contribute to the protective effects of these fruits against cardiovascular diseases and cancer. The metabolism of naringin (naringenin 7-O-neohesperidoside) is studied here in healthy (sham-operated, ShO) and tumor-bearing (TuB) rats. The tumor was induced by implanting Yoshida's sarcoma in hindlimb. Both groups received for 7 days a semi-synthetic diet containing 0.5% naringin in per feeding conditions. Flavanones were analyzed in plasma, liver, kidney and urine by tandem mass spectrometry. Naringenin conjugates (essentially glucuronides) accounted for up to 98% of the total flavanones in plasma. Low amounts of hesperetin (4'-O-methyl naringénine) and isosakuranetin (3'-hydroxy-4'-O-methylnaringenin) were also detected in all biological samples and accounted for 2% of the total flavanones in plasma. They were largely present as aglycones. The in vivo hydroxylation of flavanones is described here for the first time. Total concentrations of naringenin metabolites reached 17.3+/-2.7 microM in plasma 6 hours after the beginning of the meal in healthy rats and only 10.6+/-1.3 microM in TuB rats. The nature of metabolites was similar in both healthy and TuB rats and in plasma, tissues and urine. The lower concentration of flavanones in the TuB rats suggests that disease and more particularly cancer, may affect the bioavailability of flavonoids.

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.

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.

Transport of proanthocyanidin dimer, trimer, and polymer across monolayers of human intestinal epithelial Caco-2 cells.

The gut absorption of proanthocyanidins (PAs) and of the related (+)-catechin monomer was investigated with colonic carcinoma (Caco-2) cells of a human origin, grown in monolayers on permeable filters. Permeability of various radiolabeled PAs differing in their molecular weight was compared with that of the radiolabeled (+)-catechin. No toxicity was observed at PA concentrations up to the physiological concentration of 1 mM. (+)-Catechin and PA dimer and trimer had similar permeability coefficients (P(app) = 0.9-2.0 x 10(-6) cm s(-1)) close to that of mannitol, a marker of paracellular transport. Paracellular transport was also indicated by the increase of absorption after reduction of the transepithelial electric resistance through calcium ion removal. In contrast, permeability of a PA polymer with an average polymerization degree of 6 (molecular weight 1,740) was approximately 10 times lower (P(app) = 0.10 +/- 0.04 x 10(-6) cm s(-1)). PAs, particularly the most astringent PA polymer, were also adsorbed on the epithelial cells. These results suggest that PA dimers and trimers could be absorbed in vivo and that polymer bioavailability is limited to the gut lumen.

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.

Polymeric proanthocyanidins are catabolized by human colonic microflora into low-molecular-weight phenolic acids.

Polymeric proanthocyanidins are common constituents of many foods and beverages. Their fate in the human body remains largely unknown. Their metabolism by human colonic microflora incubated in vitro in anoxic conditions has been investigated using nonlabeled and (14)C-labeled purified proanthocyanidin polymers. Polymers were almost totally degraded after 48 h of incubation. Phenylacetic, phenylpropionic and phenylvaleric acids, monohydroxylated mainly in the meta or para position, were identified as metabolites by gas chromatography coupled to mass spectrometry (GC-MS). Yields were similar to those previously reported for flavonoid monomers. These results provide the first evidence of degradation of dietary phenolic polymers into low-molecular-weight aromatic compounds. To understand the nutritional properties of proanthocyanidins, it is therefore essential to consider the biological properties of these metabolites.