Metabolism of antioxidant and chemopreventive ellagitannins from strawberries, raspberries, walnuts, and oak-aged wine in humans: identification of biomarkers and individual variability

Ellagitannins (ETs) are dietary polyphenols, containing ellagic acid (EA) subunits, with antioxidant and cancer chemopreventive activities that might contribute to health benefits in humans. However, little is known about their metabolic fate. We investigate here the metabolism of different dietary ETs and EA derivatives in humans. Forty healthy volunteers were distributed in four groups. Each group consumed, in a single dose, a different ET-containing foodstuff, i.e., strawberries (250 g), red raspberries (225 g), walnuts (35 g), and oak-aged red wine (300 mL). After the intake, five urine fractions (F) were collected at 8 (F1), 16 (F2), 32 (F3), 40 (F4), and 56 (F5) h. Neither ETs nor EA were detected in urine after LC-MS/MS analysis. However, the microbial metabolite 3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one (urolithin B) conjugated with glucuronic acid was detected along the fractions F3-F5 in all of the subjects, independently of the consumed foodstuff. The mean percentage of metabolite excretion ranged from 2.8 (strawberries) to 16.6% (walnuts) regarding the ingested ETs. Considerable interindividual differences were noted, identifying "high and low metabolite excreters" in each group, which supported the involvement of the colonic microflora in ET metabolism. These results indicate that urolithin B (a previously described antiangiogenic and hyaluronidase inhibitor compound) is a biomarker of human exposure to dietary ETs and may be useful in intervention studies with ET-containing products. The antioxidant and anticarcinogenic effects of dietary ETs and EA should be considered in the gastrointestinal tract whereas the study of potential systemic activities should be focused on the bioavailable urolithin B derivatives.     

Ingestion of oregano extract increases excretion of urinary phenolic metabolites in humans

Despite the promising antioxidant action of Lamiaceae herbs in vitro, human studies on these potential sources of dietary antioxidants have remained scarce. In this work, the phenolic acids recovered in human urine after single ingestion of Origanum onites extract were analyzed. The excretion was increased 4- and 2-fold during 0-24 and 24-48 h of the follow-up, respectively. The mean increase in the excretion of phenolic compounds exceeded the ingested amount of identified phenolic acids. The result can be partly explained by rosmarinic acid, the main identified phenolic constituent in the extract, as well as flavonoids present in minor amounts, presumably being metabolized into a double amount of simple phenolic metabolites. Furthermore, unidentified phenolic constituents in the extract partly contribute to the excretory increase. The main metabolite, p-hydroxybenzoic acid, was excreted rapidly. The results show that constituents of oregano extract and, in particular, their metabolites may contribute to the dietary intake of phenolic antioxidants.     

Urinary and plasma levels of resveratrol and quercetin in humans, mice, and rats after ingestion of pure compounds and grape juice

The present study investigates the bioavailability of resveratrol and quercetin in humans, mice, and rats after oral ingestion of grape juice preparations or pure aglycones. Oral administration of resveratrol and quercetin to humans yielded detectable levels of resveratrol, quercetin, and their derivatives in the plasma and urine. Urinary levels of resveratrol, quercetin, and their metabolites were observed in human subjects receiving 600 and 1200 mL of grape juice, whereas quercetin metabolites were identified in urine samples even after receiving 200 mL of grape juice. The cumulative amounts of resveratrol and quercetin excreted in the urine of mice receiving concentrated grape juice for 4 days were 2.3 and 0.7% of the ingested doses, respectively. After i.g. administration of resveratrol to rats (2 mg/kg), up to 1.2 microM resveratrol was observed in the plasma. The study demonstrates that the glycoside forms of resveratrol and quercetin in grape juice are absorbed to a lesser extent than the aglycones.     

A metabonomic strategy for the detection of the metabolic effects of chamomile (Matricaria recutita L.) ingestion

A metabonomic strategy, utilizing high-resolution 1H NMR spectroscopy in conjunction with chemometric methods (discriminant analysis with orthogonal signal correction), has been applied to the study of human biological responses to chamomile tea ingestion. Daily urine samples were collected from volunteers during a 6-week period incorporating a 2-week baseline period, 2 weeks of daily chamomile tea ingestion, and a 2-week post-treatment phase. Although strong intersubject variation in metabolite profiles was observed, clear differentiation between the samples obtained before and after chamomile ingestion was achieved on the basis of increased urinary excretion of hippurate and glycine with depleted creatinine concentration. Samples obtained up to 2 weeks after daily chamomile intake formed an isolated cluster in the discriminant analysis map, from which it was inferred that the metabolic effects of chamomile ingestion were prolonged during the 2-week postdosing period. This study highlights the potential for metabonomic technology in the assessment of nutritional interventions, despite the high degree of variation from genetic and environmental sources.     

Methyleugenol in Ocimum basilicum L. Cv. genovese gigante

Ocimum basilicum cv. Genovese Gigante is the basil cultivar used the most in the production of a typical Italian sauce called pesto. The aromatic composition of plants at different growth stages was determined. Plants from different areas of northwestern Italy were analyzed at 4 and 6 weeks after sowing and showed methyleugenol and eugenol as the main components. The content of these compounds was correlated with plant height rather than plant age. Particularly, methyleugenol was predominant in plants up to 10 cm in height, whereas eugenol was prevalent in taller plants. These results are important in the evaluation of risk to human health posed by dietary ingestion of methyleugenol contained in pesto.     

LC/ES-MS detection of hydroxycinnamates in human plasma and urine

Hydroxycinnamates are components of many fruits and vegetables, being present in particularly high concentrations in prunes. An abundance of phenolic compounds in the diet has been associated with reduced heart disease mortality. However, little is known about the absorption and metabolism of these metabolites after normal foods are consumed. An LC--electrospray--MS method was developed to measure the concentration of caffeic acid in human plasma and urine, but it can also be applied to ferulic acid and chlorogenic acid. The limit of detection was found to be 10.0 nmol/L for caffeic acid and 12.5 nmol/L for ferulic and chlorogenic acids. The method was tested on samples of plasma and urine collected from volunteers who consumed a single dose of 100 g of prunes and increased levels were observed, demonstrating that the method is capable of detecting changes in hydroxycinnamate levels induced by dietary consumption.     

Sulfated ferulic acid is the main in vivo metabolite found after short-term ingestion of free ferulic acid in rats

The bioavailability of ferulic acid (FA; 3-methoxy-4-hydroxycinnamic acid) and its metabolites was investigated in rat plasma and urine after an oral short-term ingestion of 5.15 mg/kg of FA. Free FA, glucuronoconjugates, and sulfoconjugates were quickly detected in plasma with a peak of concentration found 30 min after ingestion. Sulfoconjugates were the main derivates ( approximately 50%). In urine, the cumulative excretion of total metabolites reached a plateau 1.5 h after ingestion, and approximately 40% were excreted by this way. Free FA recovered in urine represented only 4.9 +/-1.5% of the native FA consumed by rats. Glucuronoconjugates and sulfoconjugates represented 0.5 +/- 0.3 and 32.7 +/- 7.3%, respectively. These results suggested that a part of FA incorporated in the diet was quickly absorbed and largely metabolized in sulfoconjugates before excretion in urine.     

Validation of a new LC-MS/MS method for the detection and quantification of phenolic metabolites from tomato sauce in biological samples

Tomato is a good source of bioactive molecules such as vitamin C, carotenoids, and phenolic compounds. Up to now, only a few studies have evaluated the bioavailability of phenolic compounds from tomato. This paper presents the optimization of a method for the determination of phenolics in tomato and their metabolites in human urine and plasma after ingestion of tomato sauce. The sample preparation includes a SPE step to obtain cleaner extracts for injection in the LC-MS/MS system. The mean recovery of analytes ranged from 73 to 104% in plasma and from 65 to 106% in urine, the accuracy was between 90.3 and 115.0% in urine and between 85.7 and 115.0% in plasma, and the precision coefficient of variation was <15%. The method allowed detection and quantification limits of 0.5-29 and 2.0-90 ng mL?1 in urine, respectively, and 0.5-30 and 2.0-105 ng mL?1 in plasma, respectively, for the same phenolic compounds.     

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.     

Formation of N-nitroso-N-methylurea in various samples of smoked/dried fish, fish sauce, seafoods, and ethnic fermented/pickled vegetables following incubation with nitrite under acidic conditions

In continuation of our previous studies on N-nitroso-N-methylurea (NMU) formation in cured meats following incubation with nitrite at gastric pH, we extended the investigation to other foods mentioned in the title of this paper. The main objective was to determine whether these foods have the potential to form NMU at pH's that can be found in the human stomach. This was done by nitrosating an aliquot (5 g for fish sauce, 10 g for the others) of each with 7.25 microM to 1.59 mM levels of sodium nitrite for 2 h at room temperature at pH 0.8--1.5 and measuring the amounts of NMU formed. Of the samples tested, fish sauce formed 2--712 ng of NMU, followed in decreasing order by herring (<0.3--688 ng); dried anchovy, shrimp, and other fishes (<0.3--134 ng); crab and lobster paté (<0.3--342 ng); sardines (6--59 ng); oysters and mussels (11--31 ng); dried squid (3--47 ng); kimchi (7--107 ng); and Japanese pickled radish (<0.3--72 ng). Incorporation of 200-2000 ppm of ascorbic acid in the fish sauce and other foods, prior to nitrosation, appreciably inhibited such NMU formation. Although previous researchers in China reported NMU formation in nitrosated samples of fish sauce, this is the first reported formation of NMU upon nitrosation of the other foods mentioned above, and the first reported inhibition of such formation by added ascorbic acid.     

Monitoring acetaldehyde concentrations during micro-oxygenation of red wine by headspace solid-phase microextraction with on-fiber derivatization

An analytical method was developed to quantify levels of acetaldehyde in wine samples. The method utilizes headspace solid-phase microextraction with on-fiber derivatization using O-(pentafluorobenzyl)hydroxylamine and quantification by gas chromatography with flame ionization detection. The technique showed good sensitivity and reproducibility in samples of Chardonnay, Petite Sirah, and Merlot wines containing acetaldehyde at levels below the sensory threshold (40-100 ppm). The method was used to monitor acetaldehyde concentrations during the micro-oxygenation of Merlot wine in a 141 L pilot-plant experiment and a 2400 L full-scale study. In both experiments, levels of acetaldehyde remained constant for several weeks before increasing at rates of the order of 1 ppm/day. Variations in the levels of acetaldehyde present are discussed within the context of the underlying chemical reactions.     

Gluconic acid consumption in wines by Schizosaccharomyces pombe and its effect on the concentrations of major volatile compounds and polyols

Schizosaccharomyces pombe 1379 (ATCC 26760) yeast strain in wine substantially increases acetaldehyde and 1,1-diethoxyethane concentrations and to decreases levo-2,3-butanediol, glycerol, acetoin, and gluconic acid concentrations. In this study, S. pombe has been used for the first time to reduce gluconic acid in wine under aerobic conditions. Only acetaldehyde and acetoin exhibited significantly higher levels in the wines containing gluconic acid. The high in vitro specific activity of alcohol dehydrogenase observed may be directly related to the high production of acetaldehyde by the studied fission yeast.     

LC-MS/MS quantification of sulforaphane and indole-3-carbinol metabolites in human plasma and urine after dietary intake of selenium-fortified broccoli

This study aimed at developing a sensitive LC-MS/MS method for the quantification of sulforaphane (SFN) and indole-3-carbinol metabolites in plasma and urine after dietary intake of regular and selenium-fertilized broccoli using stable isotope dilution analysis. In a three-armed, placebo-controlled, randomized human intervention study with 76 healthy volunteers, 200 g of regular (485 μg of total glucosinolates and <0.01 μg of selenium per gram fresh weight) or selenium-fertilized broccoli (589 μg of total glucosinolates and 0.25 μg of selenium per gram fresh weight) was administered daily for 4 weeks. Glucoraphanin and glucobrassicin metabolites quantified in plasma and urine were SFN-glutathione, SFN-cysteine, SFN-cysteinylglycine, SFN-acetylcysteine, and indole-3-carboxaldehyde, indole-3-carboxylic acid, and ascorbigen, respectively. Dietary intake of selenium-fertilized broccoli increased serum selenium concentration analyzed by means of atomic absorption spectroscopy by up to 25% (p < 0.001), but affected neither glucosinolate concentrations in broccoli nor their metabolite concentrations in plasma and urine compared to regular broccoli.     

Interaction of yeasts with the products resulting from the condensation reaction between (+)-catechin and acetaldehyde

The condensation reaction between (+)-catechin and acetaldehyde was studied in model solutions in the presence and absence yeasts in order to evaluate its contribution to color changes in fermented drinks such as white wine. On the basis of the results, the yeasts retain the oligomers produced in the reaction, their retention ability increasing for higher polymerization degrees. As a result, the color of model solutions, measured as the absorbance at 420 nm, was found to decrease after the addition of yeasts. On the other hand, the yeasts exhibited no inhibitory effect on the condensation reaction, which took place at the same rate in their presence and absence. At acidity levels and reactant concentrations similar to those in wine, with acetaldehyde in high concentration as it is present in sherry wines, the reaction was found to occur very slowly. Taking into account that Yeasts are present during most of the winemaking process; consequently, they retain oligomers, and the studied reaction could mainly contribute to the alteration of the color of white wine after bottling.     

Cranberries and cranberry products: powerful in vitro, ex vivo, and in vivo sources of antioxidants

Cranberry products and especially cranberry juice (CJ) have been consumed for health reasons primarily due to their effect on urinary tract infections. We investigated the quantity of both free and total (after hydrolysis) phenolic antioxidants in cranberry products using the Folin assay. The order of amount of total polyphenols in cranberry foods on a fresh weight basis was as follows: dried > frozen > sauce > jellied sauce. On a serving size basis for all cranberry products, the order was as follows: frozen > 100% juice > dried > 27% juice > sauce > jellied sauce. High fructose corn syrup (HFCS) is a major source of sugar consumption in the U.S. and contains both glucose and fructose, potential mediators of oxidative stress. We investigated the effect of the consumption of HFCS and ascorbate with CJ antioxidants or without CJ (control) given to 10 normal individuals after an overnight fast. Plasma antioxidant capacity, glucose, triglycerides, and ascorbate were measured 6 times over 7 h after the consumption of a single 240 mL serving of the two different beverages. The control HFCS caused a slight decrease in plasma antioxidant capacity at all time points and thus an oxidative stress in spite of the presence of ascorbate. CJ produced an increase in plasma antioxidant capacity that was significantly greater than control HFCS at all time points. Postprandial triglycerides, due to fructose in the beverages, were mainly responsible for the oxidative stress and were significantly correlated with the oxidative stress as measured by the antioxidant capacity. Cranberries are an excellent source of high quality antioxidants and should be examined in human supplementation studies.     

Identification of food-derived collagen peptides in human blood after oral ingestion of gelatin hydrolysates

In the present study, we identified several food-derived collagen peptides in human blood after oral ingestion of some gelatin hydrolysates. Healthy human volunteers ingested the gelatin hydrolysates (9.4-23 g) from porcine skin, chicken feet, and cartilage after 12 h of fasting. Negligible amounts of the peptide form of hydroxyproline (Hyp) were observed in human blood before the ingestion. After the oral ingestion, the peptide form of Hyp significantly increased and reached a maximum level (20-60 nmol/mL of plasma) after 1-2 h and then decreased to half of the maximum level at 4 h after the ingestion. Major constituents of food-derived collagen peptides in human serum and plasma were identified as Pro-Hyp. In addition, small but significant amounts of Ala-Hyp, Ala-Hyp-Gly, Pro-Hyp-Gly, Leu-Hyp, Ile-Hyp, and Phe-Hyp were contained.     

Akt/FOXO3a/SIRT1-mediated cardioprotection by n-tyrosol against ischemic stress in rat in vivo model of myocardial infarction: switching gears toward survival and longevity

Moderate consumption of wine has been associated with decreased risk of cardiovascular events. Recently we have shown that white wine is equally as cardioprotective as red wine. However, unlike resveratrol (polyphenol in red wine), the white wine component, n-tyrosol [2-(4-hydroxyphenyl)ethanol] has not been explored for its cardioprotective effect and mechanism of action. Therefore, the present study was designed to evaluate the effect of tyrosol treatment (5 mg/kg/day for 30 days) on myocardial ischemic stress in a rat in vivo model of Myocardial Infarction (MI) and to identify key molecular targets involved in this mechanism. MI was induced by Left Anterior Descending (LAD) coronary artery ligation. Reduced infarct size (32.42 vs 48.03%) and cardiomyocyte apoptosis (171 vs 256 counts/100 HPF) were observed along with improvement in the myocardial functional parameters such as LVIDs (5.89 vs 6.58 mm), ejection fraction (51.91 vs 45.09%), and fractional shortening (28.46 vs 23.52%) as assessed by echocardiography in the tyrosol-treated animals when compared to the nontreated controls. We have also observed significant increase in the phosphorylation of Akt (1.4-fold), eNOS (3-fold) and FOXO3a (2.6-fold). In addition, tyrosol induced the expression of longevity protein SIRT1 (3.2-fold) in the MI group as compared to the non-treated MI control. Therefore tyrosol's SIRT1, Akt and eNOS activating power adds another dimension to the white wine research, because it adds a great link to the French paradox. In conclusion these findings suggest that tyrosol induces myocardial protection against ischemia related stress by inducing survival and longevity proteins that may be considered as anti-aging therapy for the heart. However, human intervention studies would be necessary before establishing any recommendations about dietary habits for tyrosol intake or administration of dietary supplements containing tyrosol.     

Histamine behavior during the fermentation process in the manufacture of fish sauce.

The behavior of histamine in fish sauce making was investigated using fresh and spoiled fish with or without histidine added during fermentation. The histamine content in the 2% histidine added fresh fish mixture did not change significantly even after a lapse of 4 months incubation. However, when histidine was added to spoiled fish, the histamine content rose to a high level but decreased continuously with incubation time. This decrease may suggest the presence of histamine-decomposing bacteria in the samples. Eight of the 10 commercial fish sauces analyzed contained histamine levels below the "decomposition level" of 50 mg/kg set by the FDA. The increase in histamine at the initial stage and the decrease in histidine might suggest that histidine was converted to histamine by a microorganism possessing the enzyme histidine decarboxylase.     

Iberian pig as a model to clarify obscure points in the bioavailability and metabolism of ellagitannins in humans

Ellagitannin-containing foods (strawberries, walnuts, pomegranate, raspberries, oak-aged wine, etc.) have attracted attention due to their cancer chemopreventive, cardioprotective, and antioxidant effects. Ellagitannins (ETs) are not absorbed as such but are metabolized by the intestinal flora to yield urolithins (hydroxydibenzopyran-6-one derivatives). In this study, Iberian pig is used as a model to clarify human ET metabolism. Pigs were fed either cereal fodder or acorns, a rich source of ETs. Plasma, urine, bile, lumen and intestinal tissues (jejunum and colon), feces, liver, kidney, heart, brain, lung, muscle, and subcutaneous fat tissue were analyzed. The results demonstrate that acorn ETs release ellagic acid (EA) in the jejunum, then the intestinal flora metabolizes EA sequentially to yield tetrahydroxy- (urolithin D), trihydroxy- (urolithin C), dihydroxy- (urolithin A), and monohydroxy- (urolithin B) dibenzopyran-6-one metabolites, which were absorbed preferentially when their lipophilicity increased. Thirty-one ET-derived metabolites were detected, including 25 urolithin and 6 EA derivatives. Twenty-six extensively conjugated metabolites were detected in bile, glucuronides and methyl glucuronides of EA and particularly urolithin A, C, and D derivatives, confirming a very active enterohepatic circulation. Urolithins A and B as well as dimethyl-EA-glucuronide were detected in peripheral plasma. The presence of EA metabolites in bile and in urine and its absence in intestinal tissues suggested its absorption in the stomach. Urolithin A was the only metabolite detected in feces and together with its glucuronide was the most abundant metabolite in urine. No metabolites accumulated in any organ analyzed. The whole metabolism of ETs is shown for the first time, confirming previous studies in humans and explaining the long persistency of urolithin metabolites in the body mediated by an active enterohepatic circulation.     

Heterocyclic acetals from glycerol and acetaldehyde in Port wines: evolution with aging

In Port wine, isomers of glycerol and acetaldehyde acetals have been found at total contents ranging from 9.4 to 175.3 mg/L. During oxidative aging, the concentrations of the 5-hydroxy-2-methyl-1,3-dioxane and 4-hydroxymethyl-2-methyl-1,3-dioxolane isomers increased with time showing a linear correlation (r > 0.95). The flavor threshold for the mixture of the four isomers was evaluated in wine at 100 mg/L. Thus, it is expected that they contribute to "old Port wine" aroma in wines older than 30 years. Experiments with model solutions and wine clearly demonstrated that SO(2) combines with acetaldehyde and blocks the acetalization reaction.