The stereochemical configuration of flavanols influences the level and metabolism of flavanols in humans and their biological activity in vivo

Extensive epidemiological and clinical evidence associates diets high in flavanol-containing foods with cardiovascular health benefits in humans. Catechin and epicatechin, the most common flavanols in foods, are present in the diet in different enantiomeric forms. This study investigated the influence of the stereochemical configuration of flavanols on their absorption, metabolism, and biological activity. Healthy adult males were asked to consume equal amounts of the stereochemically pure flavanols (-)-epicatechin, (-)-catechin, (+)-catechin, and (+)-epicatechin (1.5mg/kg bw) in a well-defined cocoa-based, dairy-containing drink matrix, and flavanol levels were subsequently determined in plasma and 24-h urine. The results obtained show that the stereochemical configuration of flavanols has a profound influence on their uptake and metabolism in humans. In addition, we assessed the vasodilatory activity of each flavanol stereoisomer in vivo and found (-)-epicatechin to be the single stereoisomer capable of mediating a significant arterial dilation response. Importantly, this effect was independent of the classic antioxidant properties of flavanols. Overall, these results indicate that the proposed beneficial health effects associated with the consumption of flavanol-containing foods will significantly depend on the stereochemical configuration of the flavanols ingested.     

Effect of perinatally supplemented flavonoids on brain structure,circulation, cognition,and metabolism in C57BL/6J mice

Evidence suggests that flavanol consumption can beneficially affect cognition in adults, but little is known about the effect of flavanol intake early in life. The present study aims to assess the effect of dietary flavanol intake during the gestational and postnatal period on brain structure, cerebral blood flow (CBF), cognition, and brain metabolism in C57BL/6J mice.Female wild-type C57BL/6J mice were randomly assigned to either a flavanol supplemented diet or a control diet at gestational day 0. Male offspring remained on the corresponding diets throughout life and performed cognitive and behavioral tests during puberty and adulthood assessing locomotion and exploration (Phenotyper and open field), sensorimotor integration (Rotarod and prepulse inhibition), and spatial learning and memory (Morris water maze, MWM). Magnetic resonance spectroscopy and imaging at 11.7T measured brain metabolism, CBF, and white and gray matter integrity in adult mice. Biochemical and immunohistochemical analyses evaluated inflammation, synaptic plasticity, neurogenesis, and vascular density.Cognitive and behavioral tests demonstrated increased locomotion in Phenotypers during puberty after flavanol supplementation (p = 0.041) but not in adulthood. Rotarod and prepulse inhibition demonstrated no differences in sensorimotor integration. Flavanols altered spatial learning in the MWM in adulthood (p = 0.039), while spatial memory remained unaffected. Additionally, flavanols increased diffusion coherence in the visual cortex (p = 0.014) and possibly the corpus callosum (p = 0.066) in adulthood. Mean diffusion remained unaffected, a finding that corresponds with our immunohistochemical data showing no effect on neurogenesis, synaptic plasticity, and vascular density. However, flavanols decreased CBF in the cortex (p = 0.001) and thalamus (p = 0.009) in adulthood. Brain metabolite levels and neuroinflammation remained unaffected by flavanols.These data suggest that dietary flavanols results in subtle alterations in brain structure, locomotor activity and spatial learning. Comparison of these data to published findings in aging or neurodegeneration suggests that benefits of dietary flavanols may increase with advancing age and in disease.     

Epicatechin and catechin are O-methylated and glucuronidated in the small intestine

There is considerable interest in the bioavailability of polyphenols and their bioactivity in vivo. We have studied the absorption and metabolism of catechin and epicatechin in the small intestine and the comparative transfer across the jejunum and ileum. Perfusion of isolated jejunum with the flavanols resulted in glucuronidation ( approximately 45%), O-methylation: 3'-O-Methyl- and 4'-O-methyl- ( approximately 30%), and O-methyl-glucuronidation ( approximately 20% of total flavanols identified) during transfer across the enterocytes to the serosal side. This demonstrates the activity of catechol-O-methyl transferases in the metabolism of flavanols and suggests that these metabolites and conjugates are likely to enter the portal vein. In contrast, in the case of the ileum, the majority of the flavanols appeared on the serosal side unmetabolised and the total percentage of flavanols transferred was higher than that in the jejunum ( approximately fivefold).     

Structurally related (-)-epicatechin metabolites in humans: assessment using de novo chemically synthesized authentic standards

Accumulating data suggest that diets rich in flavanols and procyanidins are beneficial for human health. In this context, there has been a great interest in elucidating the systemic levels and metabolic profiles at which these compounds occur in humans. Although recent progress has been made, there still exist considerable differences and various disagreements with regard to the mammalian metabolites of these compounds, which in turn are largely a consequence of the lack of availability of authentic standards that would allow for the directed development and validation of expedient analytical methodologies. In this study, we developed a method for the analysis of structurally related flavanol metabolites using a wide range of authentic standards. Applying this method in the context of a human dietary intervention study using comprehensively characterized and standardized flavanol- and procyanidin-containing cocoa, we were able to identify the structurally related (-)-epicatechin metabolites (SREM) postprandially extant in the systemic circulation of humans. Our results demonstrate that (-)-epicatechin-3'-β-D-glucuronide, (-)-epicatechin-3'-sulfate, and a 3'-O-methyl-(-)-epicatechin-5/7-sulfate are the predominant SREM in humans and further confirm the relevance of the stereochemical configuration in the context of flavanol metabolism. In addition, we also identified plausible causes for the previously reported discrepancies regarding flavanol metabolism, consisting, to a significant extent, of interlaboratory differences in sample preparation (enzymatic treatment and sample conditioning for HPLC analysis) and detection systems. Thus, these findings may also aid in the establishment of consensus on this topic.     

How do dietary flavanols improve vascular function? A position paper

Epidemiological and clinical studies revealed that high-flavanol diet or isolated (−)-epicatechin improves the function of the vascular endothelium, as assessed by flow-mediated dilation, through elevation of bioavailability and bioactivity of NO^•. We have demonstrated that exposure of human endothelial cells to (−)-epicatechin elevates the cellular levels of NO^• and cyclic GMP and protects against oxidative stress elicited by proinflammatory agonists. (−)-Epicatechin acts like a prodrug, since these effects involve O-methylation of the flavanol and are attributed to apocynin-like inhibition of endothelial NADPH oxidase. Thus, generation of superoxide and peroxynitrite is diminished and, consequently, the cellular NO^• level is preserved or augmented. We propose therefore that endothelial NO^• metabolism rather than general antioxidant activity is a major target of dietary flavanols and that NADPH oxidase activity is a crucial site of action. Moreover, flavonoid glucuronides appear to serve as plasma transport metabolites to target cells rather than solely as excretion products. Implications for the interpretation of the role of dietary polyphenols for cardiovascular health are discussed.     

Food effects on the absorption and pharmacokinetics of cocoa flavanols

Macronutrients in food and gastric acid are known to have a pronounced effect on the metabolism of many xenobiotics, an effect that impacts their efficacy as bioactive agents. In this investigation we assessed the impact of select food treatments and the histamine H(2)-receptor antagonist Famotidine (Pepcid-AC) on flavanol absorption and metabolism. Four crossover intervention studies were conducted with 6 subjects each. Volunteers consumed sugar-free, flavanol-rich cocoa (0.125 g/kg body wt) alone, with macronutrient-rich foods (8.75 or 17.5 kJ/kg subject body wt) or Famotidine (Pepcid-AC). Blood samples were drawn at 5 time points including baseline. Plasma samples were analyzed for epicatechin and catechin flavanols by HPLC. Pharmacokinetic parameters were assessed using non-compartmental methodology. When provided at 17.5 kJ/kg subject body weight (approximately 4 kcal/kg), sugar and bread test meals increased flavanol area under the curve (AUC) values to 140% of control values (P < 0.05). A corresponding tendency for plasma antioxidant capacity to increase was observed for the cocoa treatment at 1.5 and 2.5 h (P < 0.17, P < 0.06, respectively). The ability of treatment meals to affect AUC values was positively correlated with treatment carbohydrate content (r = 0.83; P< 0.02). In contrast to carbohydrate rich meals, lipid and protein rich meals and Famotidine treatment had minimal effects on flavanol absorption. Based on C(max) and AUC values, this data suggests that the uptake of flavanols can be increased significantly by concurrent carbohydrate consumption.     

Flavonoid metabolism: the interaction of metabolites and gut microbiota

Abstract

Several dietary flavonoids exhibit anti-oxidative, anti-inflammatory, and anti-osteoporotic activities relevant to prevention of chronic diseases, including lifestyle-related diseases. Dietary flavonoids (glycoside forms) are enzymatically hydrolyzed and absorbed in the intestine, and are conjugated to their glucuronide/sulfate forms by phase II enzymes in epithelial cells and the liver. The intestinal microbiota plays an important role in the metabolism of flavonoids found in foods. Some specific products of bacterial transformation, such as ring-fission products and reduced metabolites, exhibit enhanced properties. Studies on the metabolism of flavonoids by the intestinal microbiota are crucial for understanding the role of these compounds and their impact on our health. This review focused on the metabolic pathways, bioavailability, and physiological role of flavonoids, especially metabolites of quercetin and isoflavone produced by the intestinal microbiota.     

Bioavailability of isoflavones

Isoflavones are disease protective components of soybeans. Isoflavone metabolism and bioavailability are key to understanding their biological effects. Isoflavone glucuronides, dominant biotransformation products in humans that are more hydrophilic than isoflavone aglycones, activate human natural killer cells in vitro but are less toxic to NK cells than the parent aglycones. Gut microbial isoflavone metabolites have also been identified, but remain to be well characterized. Gut transit time (GTT) seems to be a significant determinant of isoflavone bioavailability because women with more rapid GTT (<40 h) experienced 2-3-fold greater absorption of isoflavones than did women with longer GTT (>65 h). Isoflavone metabolism varies a great deal among individuals, thus limiting the quantitative value of urine or plasma isoflavones as biomarkers of soy ingestion. Defining and lessening interindividual variation in isoflavone bioavailability, and characterizing health-related effects of key isoflavone metabolites are likely to be crucial to further understanding of the health benefits of isoflavones.     

Interactions between cocoa flavanols and inorganic nitrate: Additive effects on endothelial function at achievable dietary amounts

Dietary intervention studies have shown that flavanols and inorganic nitrate can improve vascular function, suggesting that these two bioactives may be responsible for beneficial health effects of diets rich in fruits and vegetables. We aimed to study interactions between cocoa flavanols (CF) and nitrate, focusing on absorption, bioavailability, excretion, and efficacy to increase endothelial function. In a double-blind randomized, dose–response crossover study, flow-mediated dilation (FMD) was measured in 15 healthy subjects before and at 1, 2, 3, and 4 h after consumption of CF (1.4–10.9 mg/kg bw) or nitrate (0.1–10 mg/kg bw). To study flavanol–nitrate interactions, an additional intervention trial was performed with nitrate and CF taken in sequence at low and high amounts. FMD was measured before (0 h) and at 1 h after ingestion of nitrate (3 or 8.5 mg/kg bw) or water. Then subjects received a CF drink (2.7 or 10.9 mg/kg bw) or a micro- and macronutrient-matched CF-free drink. FMD was measured at 1, 2, and 4 h thereafter. Blood and urine samples were collected and assessed for CF and nitric oxide (NO) metabolites with HPLC and gas-phase reductive chemiluminescence. Finally, intragastric formation of NO after CF and nitrate consumption was investigated. Both CF and nitrate induced similar intake-dependent increases in FMD. Maximal values were achieved at 1 h postingestion and gradually decreased to reach baseline values at 4 h. These effects were additive at low intake levels, whereas CF did not further increase FMD after high nitrate intake. Nitrate did not affect flavanol absorption, bioavailability, or excretion, but CF enhanced nitrate-related gastric NO formation and attenuated the increase in plasma nitrite after nitrate intake. Both flavanols and inorganic nitrate can improve endothelial function in healthy subjects at intake amounts that are achievable with a normal diet. Even low dietary intake of these bioactives may exert relevant effects on endothelial function when ingested together.     

Dopamine and Serotonin Metabolism in Brain Sites Ipsi- and Contralateral to Direction of Conditioned Turning in Rats

Concentrations of dopamine, serotonin, and some of their metabolites were analyzed by means of HPLC in brain samples obtained from rats operantly conditioned to turn in circles to obtain water reinforcement. In experiment 1 using Wistar rats, no differences in the levels of transmitters or metabolites were detected between brain samples (frontal cortex, ventral striatum, dorsal striatum, septum, amygdala, substantia nigra) from the hemispheres located ipsi- and contralateral to the direction of turning. A higher dopamine metabolism (indicated by higher metabolite/transmitter ratios) in ventral striatum, dorsal striatum, and amygdala was found after 15 min than after 5 min of turning in both hemispheres. A higher dopamine metabolism was found in water-deprived rats compared to nondeprived rats independently of whether or not deprived rats were trained to turn for water reinforcement. In two additional experiments, no differences in dopamine metabolism were found between the ipsi- and contralateral striatum of Wistar rats after 25 min and Sprague-Dawley rats after 10 min of operantly conditioned turning. The present results confirm that dopamine metabolism can change with different behavioral or physiological states; they do not support the hypothesis that conditioned turning is correlated with asymmetrical changes in the metabolism of dopamine or serotonin in the brain.     

Physiological variation in plasma total homocysteine concentrations in rats

Hyperhomocysteinemia was initially related to cardiovascular diseases; but homocysteine (Hcy) metabolism disturbances have more recently associated with a wide range of pathophysiological conditions including age-related diseases, disrupted circadian rhythms and gynaecological disorders. Since in many cases we do not know to what extent animal models are physiologically similar to human ones, this study aimed to track spontaneous variations in rat plasma Hcy concentrations during different physiological processes such as life cycle, 24 hours and estrous cycle. Plasma total Hcy concentrations were accessed by HPLC. Plasma Hcy concentration varied with age and newborns had the lowest values (2.94 +/- 0.47 micromol/L). Rats aged 10 days presented concentration similar to 3 month old animals (6.87 +/- 0.67 and 8.29 +/- 1.55 micromol/L respectively). Values decreased to 6.42 +/- 1.65 micromol/L at 6 months and 4.87 +/- 0.81 micromol/L at 28 months. Concerning circadian variations in Hcy concentration cosinor analysis showed acrophase in young rats at 1:09 pm, but no plasma Hcy circadian variations in aged rats. Female rats showed changes in Hcy concentration during the estrous cycle with higher values during the diestrous I (10.61 +/- 1.81 micromol/L) compared with the estrous (8.47 +/- 1.86 micromol/L) and diestrous II (7.68 +/- 1.58 micromol/L) phases. In conclusion, plasma Hcy concentration varied spontaneously with ontogenic development and during the estrous cycle and presented a circadian rhythm variation in young rats.     

Selenium metabolism in rats after administration of toxic doses of selenite

We studied organ concentration, excretion and excreted forms of selenium in young and adult rats after a single s.c. injection of a sublethal dose of 75Se-selenite. In the young about a 10-fold higher concentration of 75Se in blood, liver, kidneys and In the young, about a 10-fold higher concentration of 75Se in blood, liver, kidneys and heart was found at all the experimental intervals studied (1-7 days). The highest 75Se concentration in the young was in the liver while in the adults it was found in the kidneys. The spectrum of radioselenium metabolites in the urine was the same in both groups. However, the main product excreted by young rats was 75Se-glutathione selenotrisulphide and an unidentified neutral substance while it was the trimethylselenonium ion in the adults. Ontogenetic differences in selenium metabolism could be one of the factors underlying the differences in the response of the young and the adult rats to toxic doses of selenite.     

Metabonomic alterations in hippocampus, temporal and prefrontal cortex with age in rats

The metabolic changes in hippocampus, temporal cortex and prefrontal cortex in SD rats along with aging were explored using a metabonomic approach, which based on high resolution “magic angle spinning” ¹H NMR spectroscopy. The metabolite profiles were analyzed by partial least squares-discriminant analysis, and the results showed that the metabolites of the above three brain regions in old rats were dramatically different from that in the adult and young rats. The old rats showed increased myo-inositol and lactate in all of the three brain regions, and decreased N-acetylaspartate in temporal and frontal cortex, Glutamate–GABA level became imbalance in temporal cortex of old rats. In addition, compared with the adult female rats, male rats had higher levels of N-acetylaspartate, taurine, and creatine in temporal or frontal cortex. The age-related metabolic changes may indicate the early functional alterations of neural cells in these brain regions, especially the temporal cortex. The gender-related metabolic changes suggest the significance of the hormonal regulation in brain metabolism. Our work highlights the potential of metabolic profiling to enhance our understanding of biological mechanisms of brain aging.     

Opposite effect of methionine-supplemented diet, a model of hyperhomocysteinemia, on plasma and liver antioxidant status in normotensive and spontaneously hypertensive rats

Hyperhomocysteinemia is often associated with an increase in blood pressure. However our previous study has shown that methionine supplementation induced an increase in blood pressure in Wistar-Kyoto (WKY) rats and a decrease in blood pressure in spontaneously hypertensive rats (SHR) with significant differences in plasma homocysteine (Hcy) metabolites levels. Previously liver antioxidant status has been shown to be decreased in SHR compared to WKY rats. It has been suggested that oxidative stress may predispose to a decrease in NO bioavailability and induce the flux of Hcy through the liver transsulfuration pathway. Thus the aim of this study was 1) to investigate the effect of methionine supplementation on NO-derived metabolites in plasma and urine 2) to investigate whether abnormalities in Hcy metabolism may be responsible for the discrepancies observed between WKY rats and SHR concerning blood pressure and 3) to investigate whether a methionine-enriched diet, differently modified plasma and liver antioxidant status in WKY rats an SHR. We conclude that the increase in blood pressure in WKY rats is related to high plasma cysteine levels and is not due to a decrease in NO bioavailability and that the decrease in blood pressure in SHR is associated with high plasma GSH levels after methionine supplementation. So GSH synthesis appears to be stimulated by liver oxidative stress and GSH is redistributed into blood in SHR. So the great GSH synthesis can be rationalized as an autocorrective response that leads to a decreased blood pressure in SHR.     

The Nature and the Origin of Polyphenols in Hoji-cha (Roasted Green Tea)

The nature and the origin of major polyphenols in Hoji-cha were elucidated from the investigation on the effects of heating on individual flavanols by using paperchromatographic technique. Furthermore, the direction of thermal transformation of each flavanol, and the quantitative changes of major product and original flavanol were also shown. The heating of flavanol solution resulted in epimerization, polymerization and decomposition, though the aspect of change was different with the kind of flavanol and thermal condition.     

Blood metabolite levels in normal and handicapped pied flycatchers rearing broods of different sizes

We measured levels of select metabolites (glucose, triglycerides, free fatty acids, glycerol, uric acid) and corticosterone in the blood plasma of adult pied flycatchers Ficedula hypoleuca while they were rearing broods whose sizes were modified experimentally. We also made it more difficult than normal for some pairs of birds to forage by removing certain wing and tail feathers (handicapping them). Both procedures have been shown previously to change parental workload. We did this in order to determine if the birds alter their use of nutrients in response to differences in their workload. Metabolite levels were not influenced by handicapping or brood size. However, the concentration of free fatty acids in the plasma of females and of triglycerides in the plasma of males was directly related to the frequency with which the adults fed their nestlings. These findings suggest that the two sexes have different ways of coping with the work associated with rearing the brood: females apparently undergo brief daily fasts while feeding their chicks, whereas males take more time to feed themselves while providing food for their young, and spend more time doing so as their workload increases. The flycatchers exhibited high concentrations of uric acid and corticosterone in the blood plasma; corticosterone and glycerol were positively correlated in females; and corticosterone and triglyceride levels were negatively correlated in males; all of which suggest that gluconeogenesis provides some of the energy required for their parental activities.     

Orally administered rosmarinic acid is present as the conjugated and/or methylated forms in plasma, and is degraded and metabolized to conjugated forms of caffeic acid, ferulic acid and m-coumaric acid

Rosmarinic acid (RA) is contained in various Lamiaceae herbs used commonly as culinary herbs. Although RA has various potent physiological actions, little is known on its bioavailability. We therefore investigated the absorption and metabolism of orally administered RA in rats. After being deprived of food for 12 h, RA (50 mg/kg body weight) or deionized water was administered orally to rats. Blood samples were collected from a cannula inserted in the femoral artery before and at designated time intervals after administration of RA. Urine excreted within 0 to 8 h and 8 to 18 h post-administration was also collected. RA and its related metabolites in plasma and urine were measured by LC-MS after treatment with sulfatase and/or beta-glucuronidase. RA, mono-methylated RA (methyl-RA) and m-coumaric acid (COA) were detected in plasma, with peak concentrations being reached at 0.5, 1 and 8 h after RA administration, respectively. RA, methyl-RA, caffeic acid (CAA), ferulic acid (FA) and COA were detected in urine after RA administration. These components in plasma and urine were present predominantly as conjugated forms such as glucuronide or sulfate. The percentage of the original oral dose of RA excreted in the urine within 18 h of administration as free and conjugated forms was 0.44 +/- 0.21% for RA, 1.60 +/- 0.74% for methyl-RA, 1.06 +/- 0.35% for CAA, 1.70 +/- 0.45% for FA and 0.67 +/- 0.29% for COA. Approximately 83% of the total amount of these metabolites was excreted in the period 8 to 18 h after RA administration. These results suggest that RA was absorbed and metabolized as conjugated and/or methylated forms, and that the majority of RA absorbed was degraded into conjugated and/or methylated forms of CAA, FA and COA before being excreted gradually in the urine.     

Cellular uptake and metabolism of flavonoids and their metabolites: implications for their bioactivity

Flavonoids have been proposed to act as beneficial agents in a multitude of disease states, including cancer, cardiovascular disease, and neurodegenerative disorders. The biological effect of these polyphenols and their in vivo circulating metabolites will ultimately depend on the extent to which they associate with cells, either by interactions at the membrane or more importantly their uptake. This review summarises the current knowledge on the cellular uptake of flavonoids and their metabolites with particular relevance to further intracellular metabolism and the generation of potential new bioactive forms. Uptake and metabolism of the circulating forms of flavanols, flavonols, and flavanones into cells of the skin, the brain, and cancer cells is reviewed and potential biological relevance to intracellular formed metabolites is discussed.