Comparison of chemometric strategies for potential exposure marker discovery and false-positive reduction in untargeted metabolomics: application to the serum analysis by LC-HRMS after intake of Vaccinium fruit supplements.

Untargeted liquid chromatographic-high-resolution mass spectrometric (LC-HRMS) metabolomics for potential exposure marker (PEM) discovery in nutrikinetic studies generates complex outputs. The correct selection of statistically significant PEMs is a crucial analytical step for understanding nutrition-health interactions. Hence, in this paper, different chemometric selection workflows for PEM discovery, using multivariate or univariate parametric or non-parametric data analyses, were comparatively tested and evaluated. The PEM selection protocols were applied to a small-sample-size untargeted LC-HRMS study of a longitudinal set of serum samples from 20 volunteers after a single intake of (poly)phenolic-rich Vaccinium myrtillus and Vaccinium corymbosum supplements. The non-parametric Games-Howell test identified a restricted group of significant features, thus minimizing the risk of false-positive retention. Among the forty-seven PEMs exhibiting a statistically significant postprandial kinetics, twelve were successfully annotated as purine pathway metabolites, benzoic and benzodiol metabolites, indole alkaloids, and organic and fatty acids, and five (i.e. octahydro-methyl-ß-carboline-dicarboxylic acid, tetrahydro-methyl-ß-carboline-dicarboxylic acid, citric acid, caprylic acid, and azelaic acid) were associated to Vaccinium berry consumption for the first time. The analysis of the area under the curve of the longitudinal dataset highlighted thirteen statistically significant PEMs discriminating the two interventions, including four intra-intervention relevant metabolites (i.e. abscisic acid glucuronide, catechol sulphate, methyl-catechol sulphate, and α-hydroxy-hippuric acid). Principal component analysis and sample classification through linear discriminant analysis performed on PEM maximum intensity confirmed the discriminating role of these PEMs.

Black soybean improves the vascular function through an increase in nitric oxide and a decrease in oxidative stress in healthy women.

Vascular dysfunction and injurious stimuli such as oxidative stress is closely related to the risk of cardiovascular diseases (CVD). Dietary polyphenols is reported to exert the beneficial effects on reducing the risk of CVD. Black soybean is rich in polyphenols, including isoflavones, anthocyanidins and flavan-3-ols, and its prevention effects on CVD risk were reported in the animal experiments. In this study, we investigated the effect of black soybean consumption on the vascular function and oxidative stress associating with the polyphenol concentrations in healthy women. Lowered vascular age was observed in 33 out of 44 volunteers who completed the 8-week trial. It was observed that improvement of the vascular stiffness, increasing in the urinary NO2 and NO3 level, and decreasing in the oxidative stress markers, 8-hydroxy-2'-deoxyguanosine, hexanoyl-lysine and myeloperoxidase. In addition, concentration of 12 polyphenols in black soybean increased in the plasma and urine. Increased concentration of polyphenols would be involved in the decreased oxidative stress. Thus, black soybean consumption improved the vascular function through an increase in nitric oxide and a decrease in oxidative stress accompanied by increasing the polyphenol concentrations in healthy women.

Pharmacokinetic Characterization of (Poly)phenolic Metabolites in Human Plasma and Urine after Acute and Short-Term Daily Consumption of Mango Pulp.

Pharmacokinetic (PK) evaluation of polyphenolic metabolites over 24 h was conducted in human subjects (n = 13, BMI = 22.7 ± 0.4 kg/m2) after acute mango pulp (MP), vitamin C (VC) or MP + VC test beverage intake and after 14 days of MP beverage intake. Plasma and urine samples were collected at different time intervals and analyzed using targeted and non-targeted mass spectrometry. The maximum concentrations (Cmax) of gallotannin metabolites were significantly increased (p < 0.05) after acute MP beverage intake compared to VC beverage alone. MP + VC beverage non-significantly enhanced the Cmax of gallic acid metabolites compared to MP beverage alone. Pyrogallol (microbial-derived metabolite) derivatives increased (3.6%) after the 14 days of MP beverage intake compared to 24 h acute MP beverage intake (p < 0.05). These results indicate extensive absorption and breakdown of gallotannins to galloyl and other (poly)phenolic metabolites after MP consumption, suggesting modulation and/or acclimation of gut microbiota to daily MP intake.

Inter-individual variability in the production of flavan-3-ol colonic metabolites: preliminary elucidation of urinary metabotypes.

PURPOSE: There is much information on the bioavailability of (poly)phenolic compounds following acute intake of various foods. However, there are only limited data on the effects of repeated and combined exposure to specific (poly)phenol food sources and the inter-individual variability in their bioavailability. This study evaluated the combined urinary excretion of (poly)phenols from green tea and coffee following daily consumption by healthy subjects in free-living conditions. The inter-individual variability in the production of phenolic metabolites was also investigated. METHODS: Eleven participants consumed both tablets of green tea and green coffee bean extracts daily for 8 weeks and 24-h urine was collected on five different occasions. The urinary profile of phenolic metabolites and a set of multivariate statistical tests were used to investigate the putative existence of characteristic metabotypes in the production of flavan-3-ol microbial metabolites. RESULTS: (Poly)phenolic compounds in the green tea and green coffee bean extracts were absorbed and excreted after simultaneous consumption, with green tea resulting in more inter-individual variability in urinary excretion of phenolic metabolites. Three metabotypes in the production of flavan-3-ol microbial metabolites were tentatively defined, characterized by the excretion of different amounts of trihydroxyphenyl-γ-valerolactones, dihydroxyphenyl-γ-valerolactones, and hydroxyphenylpropionic acids. CONCLUSIONS: The selective production of microbiota-derived metabolites from flavan-3-ols and the putative existence of characteristic metabotypes in their production represent an important development in the study of the bioavailability of plant bioactives. These observations will contribute to better understand the health effects and individual differences associated with consumption of flavan-3-ols, arguably the main class of flavonoids in the human diet.

Full 1 H and 13 C NMR spectral assignment of conjugated valerolactone metabolites isolated from urine of black tea consumers by means of SPE-prepLC-MS-LC-MS-NMR.

The health benefits of black tea have been linked to polyphenol metabolites that target specific modes of action in the human body. A major bottleneck in unravelling the underlying mechanisms is the preparative isolation of these metabolites, which hampers their structural elucidation and assessment of in vitro bioactivity. A solid phase extraction (SPE)-preparative liquid chromatography (prepLC)-MS-LC-MS-NMR workflow was implemented for preparative isolation of conjugated valerolactone metabolites of catechin-based polyphenols from urine of black tea consumers. First, the urine was cleaned and preconcentrated using an SPE method. Subsequently, the clean urine concentrate was injected on a preparative LC column, and conjugated valerolactones were obtained by MS-guided collection. Reconstituted fractions were further separated on an analytical LC column, and valerolactone fractions were collected in an MS-guided manner. These were reconstituted in methanol-d4 and identified and quantified using 1D and 2D homo- and hetereonuclear NMR experiments (at a field strength of 14.1 T), in combination with mass spectrometry. This resulted in the full spectral 1 H and 13 C NMR assignments of five conjugated valerolactones. These metabolites were collected in quantities of 8-160 µg and purities of 70-91%. The SPE-prepLC-MS-LC-MS-NMR workflow is suitable for isolating metabolites that occur at sub-µM concentrations in a complex biofluid such as urine. The workflow also provides an alternative for cumbersome and expensive de novo synthesis of tea metabolites for testing in bioactivity assays or for use as authentic analytical standards for quantification by mass spectrometry.

Bioavailability and pharmacokinetic profile of grape pomace phenolic compounds in humans.

Grape pomace, the major byproduct of the wine and juice industry, is a relevant source of bioactive phenolic compounds. However, polyphenol bioavailability in humans is not well understood, and the inter-individual variability in the production of phenolic metabolites has not been comprehensively assessed to date. The pharmacokinetic and excretive profiles of phenolic metabolites after the acute administration of a drink made from red grape pomace was here investigated in ten volunteers. A total of 35 and 28 phenolic metabolites were quantified in urine and plasma, respectively. The main circulating metabolites included phenyl-γ-valerolactones, hydroxybenzoic acids, simple phenols, hydroxyphenylpropionic acids, hydroxycinnamates, and (epi)catechin phase II conjugates. A high inter-individual variability was shown both in urine and plasma samples, and different patterns of circulating metabolites were unravelled by applying unsupervised multivariate analysis. Besides the huge variability in the production of microbial metabolites of colonic origin, an important variability was observed due to phase II conjugates. These results are of interest to further understand the potential health benefits of phenolic metabolites on individual basis.

Polyphenol intake from a Mediterranean diet decreases inflammatory biomarkers related to atherosclerosis: a substudy of the PREDIMED trial.

High dietary polyphenol intake is associated with reduced all-cause mortality and a lower incidence of cardiovascular events. However, the mechanisms involved are not fully understood. The aim of the present substudy of the PREvención con DIetaMEDiterránea (Prevention with Mediterranean diet; PREDIMED) trial was to analyse the relationship between polyphenol intake measured by total urinary polyphenol excretion (TPE), and circulating inflammatory biomarkers and cardiovascular risk factors in elderly individuals. A substudy of 1139 high-risk participants was carried out within the PREDIMED trial. The subjects were randomly assigned to a low-fat control diet or to two Mediterranean diets, supplemented with either extra-virgin olive oil or nuts. Dietary intake, anthropometric data, clinical and laboratory assessments, including inflammatory biomarkers, and urinary TPE were measured at baseline and after the one-year intervention. Participants in the highest tertile of changes in urinary TPE (T3) showed significantly lower plasma levels of inflammatory biomarkers [vascular cell adhesion molecule 1 (VCAM-1) (-9.47 ng ml-1 ), intercellular adhesion molecule 1 (-14.71 ng ml-1 ), interleukin 6 (-1.21 pg ml-1 ), tumour necrosis factor alpha (-7.05 pg ml-1 ) and monocyte chemotactic protein 1 (-3.36 pg ml-1 )] than those inthe lowest tertile (T1, P < 0.02; all). A significant inverse correlation existed between urinary TPE and the plasma concentration of\VCAM-1 (r = -0.301; P < 0.001). In addition, systolic and diastolic blood pressure (BP) decreased and plasma high-density lipoprotein cholesterol increased in parallel with increasing urinary TPE (T3 vs. T1) (P < 0.005 and P = 0.004, respectively). Increases in polyphenol intake measured as urinary TPE are associated with decreased inflammatory biomarkers, suggesting a dose-dependent anti-inflammatory effect of polyphenols. In addition, high polyphenol intake improves cardiovascular risk factors- mainly BP and the lipid profile.

Bioavailability of chlorogenic acids in rats after acute ingestion of maté tea (Ilex paraguariensis) or 5-caffeoylquinic acid.

PURPOSE: Yerba maté is widely consumed in South America as different beverages, such as maté tea (roasted leaves) and chimarrão (green dried leaves), and linked to health benefits, mainly attributed to chlorogenic acids (CGAs). Health effects of CGAs depend on their bioavailability, but such data are scarce. The aim of this study was to investigate the distribution of CGAs and metabolites in tissues, hepatic and plasmatic kinetic profile and urinary excretion after ingestion of maté tea or 5-caffeoylquinic acid (5-CQA). METHODS: Wistar rats ingested maté tea (MT) or 5-CQA (ST) and were killed after 1.5 h for tissue distribution analysis (pilot study) or at 0.5, 1, 2, 4 and 8 h for liver and plasma kinetics (main experiment). Urine was collected in metabolic cages. Biological samples were analyzed by UPLC-DAD-MS with and without incubation with ß-glucuronidase and sulfatase. RESULTS: CGAs and metabolites were detected in all tissues. Caffeic acid was the main compound in plasma up to 2 h after ingestion of maté tea, while 5-CQA predominated in ST group. Concentration of microbial metabolites increased 4 h after gavage and reached higher amounts in MT plasma and liver, when compared to ST group. Approximately 4.0 % of compounds ingested by MT and 3.3 % by ST were recovered in urine up to 8 h after the gavage. CONCLUSION: The study confirms that not only absorption, but also metabolization of CGAs begins in stomach. There were differences in compounds formed from maté tea or isolated 5-CQA, showing that CGAs profile in food may influence qualitatively and quantitatively the metabolites formed in the body.

Differential Isotope Labeling of 38 Dietary Polyphenols and Their Quantification in Urine by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry.

A large number of polyphenols are consumed with the diet and may contribute to the prevention of chronic diseases such as cardiovascular diseases, diabetes, cancers, and neurodegenerative diseases. More comprehensive methods are needed to measure exposure to this complex family of bioactive plant compounds in epidemiological studies. We report here a novel method enabling the simultaneous measurement in urine of 38 polyphenols representative of the main classes and subclasses found in the diet. This method is based on differential (12)C-/(13)C-isotope labeling of polyphenols through derivatization with isotopic dansyl chloride reagents and on the analysis of the labeled polyphenols by tandem mass spectrometry. This derivatization approach overcomes the need for costly labeled standards. Different conditions for enzyme hydrolysis of polyphenol glucuronides and sulfate esters, extraction, and dansylation of unconjugated aglycones were tested and optimized. Limits of quantification varied from 0.01 to 1.1 µM depending on polyphenols. Intrabatch coefficients of variation varied between 3.9% and 9.6%. Interbatch variations were lower than 15% for 31 compounds and lower than 29% for 6 additional polyphenols out of the 38 tested. Thirty seven polyphenols were validated and then analyzed in 475, 24 h urine samples from the European Prospective Investigation on Cancer and Nutrition (EPIC) study. Thirty four polyphenols could be detected and successfully estimated and showed large interindividual variations of concentrations (2-3 orders of magnitude depending on the compound), with median concentrations spanning from 0.01 to over 1000 µM for all 34 compounds.

Identification and quantification of novel cranberry-derived plasma and urinary (poly)phenols.

Cranberries are a rich source of (poly)phenols, in particular proanthocyanidins, anthocyanins, flavonols, and phenolic acids. However, little is known about their bioavailability in humans. We investigated the absorption, metabolism, and excretion of cranberry (poly)phenols in plasma and urine of healthy young men after consumption of a cranberry juice (787 mg (poly)phenols). A total of 60 cranberry-derived phenolic metabolites were identified using UPLC-Q-TOF-MS analysis with authentic standards. These included sulfates of pyrogallol, valerolactone, benzoic acids, phenylacetic acids, glucuronides of flavonols, as well as sulfates and glucuronides of cinnamic acids. The most abundant plasma metabolites were small phenolic compounds, in particular hippuric acid, catechol-O-sulfate, 2,3-dihydroxybenzoic acid, phenylacetic acid, isoferulic acid, 4-methylcatechol-O-sulfate, α-hydroxyhippuric acid, ferulic acid 4-O-sulfate, benzoic acid, 4-hydroxyphenyl acetic acid, dihydrocaffeic acid 3-O-sulfate, and vanillic acid-4-O-sulfate. Some benzoic acids, cinnamic acids, and flavonol metabolites appeared in plasma early, at 1-2 h post-consumption. Others such as phenylacetic acids, benzaldehydes, pyrogallols, catechols, hippuric and dihydrocinnamic acid derivatives appear in plasma later (Tmax 4-22 h). The 24 h urinary recovery with respect to the amount of (poly)phenols consumed was 6.2%. Our extensive description of the bioavailability of cranberry (poly)phenols lays important groundwork necessary to start understanding the fate of these compounds in humans.

A mixed grape and blueberry extract is safe for dogs to consume.

BACKGROUND: Grape and blueberry extracts are known to protect against age-related cognitive decline. However, beneficial effects achieved by mixing grape and blueberry extracts have yet to be evaluated in dogs, or their bioavailability assessed. Of concern to us were cases of acute renal failure in dogs, after their ingestion of grapes or raisins. The European Pet Food Industry Federation (2013) considers only the grape or raisin itself to be potentially dangerous; grape-seed extracts per-se, are not considered to be a threat. Our aim was therefore to evaluate the renal and hepatic safety, and measure plasma derivatives of a polyphenol-rich extract from grape and blueberry (PEGB; from the Neurophenols Consortium) in dogs. Polyphenol expression was analyzed by UHPLC-MS/MS over 8 hours, for dogs given PEGB at 4 mg/kg. Safety was evaluated using four groups of 6 dogs. These groups received capsules containing no PEGB (control), or PEGB at 4, 20, or 40 mg/kg BW/d, for 24 weeks. Blood and urine samples were taken the week prior to study commencement, then at the end of the 24-wk study period. Routine markers of renal and liver damage, including creatinine (Creat), blood urea nitrogen, albumin, minerals, alkaline phosphatase (ALP), and alanine transaminase (ALT) were measured. Biomarkers for early renal damage were also evaluated in plasma (cystatin C (CysC), and neutrophil gelatinase-associated lipocalin (NGAL)), and urine (CysC, clusterin (Clu), and NGAL). Ratios of urinary biomarkers to Creat were calculated, and compared with acceptable maximal values obtained for healthy dogs, as reported in the literature. RESULTS: While several PEGB-specific polyphenols and metabolites were detected in dog plasma, at the end of the PEGB consumption period, our biomarker analyses presented no evidence of either renal or liver damage (Creat, BUN, ionogram, albumin and ALT, ALP). Similarly, no indication of early renal damage could be detected. Plasma CysC, urinary CysC/Creat, Clu/Creat, and NGAL/Creat ratios were all beneath reported benchmarked maximums, with no evidence of PEGB toxicity. CONCLUSIONS: Long-term consumption of a pet specific blend of a polyphenol-rich extract from grape and blueberry (PEGB; from the Neurophenols Consortium), was not associated with renal or hepatic injury, and can therefore be considered safe.

Three week dietary intervention using apricots, pomegranate juice or/and fermented sour sobya and impact on biomarkers of antioxidative activity, oxidative stress and erythrocytic glutathione transferase activity among adults.

BACKGROUND: The beneficial effects of the polyphenol (PP) rich fruits and Lactic acid bacteria fermented foods had been reported as cost-effective strategies for health promotion. Randomized controlled trial was designed to test the hypothesis that daily intake of polyphenol rich pomegranate juice (PGJ) or/ and lactic acid bacteria fermented sobya (FS) improved selected biomarkers of relevance to heath status. METHODS: The design of the human trial consisted of 35 healthy adults, who were distributed to 5 equal groups; The first group served as control and received no supplements; the second group received fresh apricot fruits (200 g); the third (PGJ) (250 g), the fourth a mixture of PGJ (150 g) and FS (140 g) and the fifth group received (FS) (170 g). The supplements were served daily between 5 - 6 pm for 21 days. Blood and urine samples were collected at days zero and 22 of the dietary intervention. The supplements were analyzed chemically for (PP) contents and total antioxidative activities and microbiologically for selected bacteria and yeast counts. The blood samples were assayed for plasma antioxidative activities and for erythrocytic glutathione transferase activity (E-GST). Urine samples were analyzed for the excretions of total PP, antioxidative activity and thiobarbituric acid reactive substances (TBARS). STATISTICAL ANALYSIS: Two way analysis of variance (ANOVA) was conducted and included the main effects of treatment, time and treatment x time interaction. RESULTS: Daily intake of (PGJ) for 3 weeks significantly increased the plasma and urinary anti-oxidative activities and reduced the urinary excretion of (TBARS). Daily intake of (FS) for 3 weeks increased only (E-GST) activity. Daily intake of a mixture of PGJ and (FS) was also effective. CONCLUSIONS: The daily intakes of PGJ and/ or (FS) affected positively selected biomarkers of relevance to health status. These functional foods have potential implication for use as bio-therapeutic foods. TRIAL REGISTRATION: The study was approved by the research ethical committee of the Ministry of Health & population, Egypt. The trial registration - the unique identifying number. (REC) decision No 12-2013-9, which complied with the Declaration of Helsinki guidelines (2004). The protocol was fully explained to all subjects and written informed consent was obtained before their participation in the trial.

Plasma and Urinary Phenolic Profiles after Acute and Repetitive Intake of Wild Blueberry.

Recent studies have shown that blueberries may have cardiovascular and cognitive health benefits. In this work, we investigated the profile of plasma and urine (poly)phenol metabolites after acute and daily consumption of wild blueberries for 30 days in 18 healthy men. The inter-individual variability in plasma and urinary polyphenol levels was also investigated. Blood samples were collected at baseline and 2 h post-consumption on day 1 and day 30. Twenty-four-hour urine was also collected on both days. A total of 61 phenolic metabolites were quantified in plasma at baseline, of which 43 increased after acute or chronic consumption of blueberries over one month. Benzoic and catechol derivatives represented more than 80% of the changes in phenolic profile after 2 h consumption on day 1, whereas hippuric and benzoic derivatives were the major compounds that increased at 0 and 2 h on day 30, respectively. The total (poly)phenol urinary excretion remained unchanged after 30 days of wild blueberry intake. The inter-individual variability ranged between 40%-48% in plasma and 47%-54% in urine. Taken together, our results illustrate that blueberry (poly)phenols are absorbed and extensively metabolized by phase II enzymes and by the gut microbiota, leading to a whole array of metabolites that may be responsible for the beneficial effects observed after blueberry consumption.

Urinary Excretion of Select Dietary Polyphenol Metabolites Is Associated with a Lower Risk of Type 2 Diabetes in Proximate but Not Remote Follow-Up in a Prospective Investigation in 2 Cohorts of US Women.

BACKGROUND: Polyphenols are phytochemicals that possess antioxidant and anti-inflammatory properties and improve glucose metabolism in animal experiments, although data from prospective epidemiologic studies examining polyphenol intakes in relation to type 2 diabetes (T2D) risk are inconsistent. OBJECTIVES: We examined urinary excretion of select flavonoid and phenolic acid metabolites, as biomarkers of intake, in relation to T2D risk. METHODS: Eight polyphenol metabolites (naringenin, hesperetin, quercetin, isorhamnetin, catechin, epicatechin, caffeic acid, and ferulic acid) were quantified in spot urine samples by liquid chromatography/mass spectrometry among 1111 T2D case-control pairs selected from the Nurses' Health Study (NHS) and NHSII. RESULTS: Higher urinary excretion of hesperetin was associated with a lower T2D risk after multivariate adjustment: the OR comparing top vs. bottom quartiles was 0.68 (95% CI: 0.49, 0.96), although a linear trend was lacking (P = 0.30). The other measured polyphenols were not significantly associated with T2D risk after multivariate adjustment. However, during the early follow-up period [≤ 4.6 y (median) since urine sample collection], markers of flavanone intakes (naringenin and hesperetin) and flavonol intakes (quercetin and isorhamnetin) were significantly associated with a lower T2D risk. The ORs (95% CIs) comparing extreme quartiles were 0.61 (0.39, 0.98; P-trend: 0.03) for total flavanones and 0.55 (0.33, 0.92; P-trend: 0.04) for total flavonols (P-interaction with follow-up length: ≤ 0.04). An inverse association was also observed for caffeic acid during early follow-up only: the OR was 0.52 (95% CI: 0.32, 0.84; P-trend: 0.03). None of these markers was associated with T2D risk during later follow-up. Metabolites of flavan-3-ols and ferulic acid were not associated with T2D risk in either period. CONCLUSIONS: These results suggest that specific flavonoid subclasses, including flavanones and flavonols, as well as caffeic acid, are associated with a lower T2D risk in relatively short-term follow-up but not during longer follow-up. Substantial within-person variability of the metabolites in single spot urine samples may limit the ability to capture associations with long-term disease risk.

Effects of total dietary polyphenols on plasma nitric oxide and blood pressure in a high cardiovascular risk cohort. The PREDIMED randomized trial.

BACKGROUND AND AIM: Hypertension is one of the main cardiovascular risk factors in the elderly. The aims of this work were to evaluate if a one-year intervention with two Mediterranean diets (Med-diet) could decrease blood pressure (BP) due to a high polyphenol consumption, and if the decrease in BP was mediated by plasma nitric oxide (NO) production. METHODS AND RESULTS: An intervention substudy of 200 participants at high cardiovascular risk was carried out within the PREDIMED trial. They were randomly assigned to a low-fat control diet or to two Med-diets, one supplemented with extra virgin olive oil (Med-EVOO) and the other with nuts (Med-nuts). Anthropometrics and clinical parameters were measured at baseline and after one year of intervention, as well as BP, plasma NO and total polyphenol excretion (TPE) in urine samples. Systolic and diastolic BP decreased significantly after a one-year dietary intervention with Med-EVOO and Med-nuts. These changes were associated with a significant increase in TPE and plasma NO. Additionally, a significant positive correlation was observed between changes in urinary TPE, a biomarker of TP intake, and in plasma NO (Beta = 4.84; 95% CI: 0.57-9.10). CONCLUSIONS: TPE in spot urine sample was positively correlated with plasma NO in Med-diets supplemented with either EVOO or nuts. The statistically significant increases in plasma NO were associated with a reduction in systolic and diastolic BP levels, adding to the growing evidence that polyphenols might protect the cardiovascular system by improving the endothelial function and enhancing endothelial synthesis of NO.

Differences in pharmacokinetics of apple polyphenols after standardized oral consumption of unprocessed apple juice.

BACKGROUND: Polyphenols are chemical compounds of the secondary plant metabolism. High concentrations can be found in various fruits including apples, berries and grapes. Polyphenols are associated with numerous health beneficial effects including a reduced risk for cardiovascular disease or diabetes. The human body cannot synthesize or store polyphenols and relies on continuous replenishment by daily diet. Unfortunately, knowledge on absorption, metabolization and excretion is still limited. The aim of this study was to determine the pharmacokinetic fate of apple polyphenols in young healthy adults. METHODS: Volunteers consumed 500 mL of an unfiltered apple juice. Blood and urine samples were collected within a time period of ten hours and analyzed for their total phenolic content, concentration of selected individual polyphenolic compounds and antioxidant capacity. RESULTS: Large differences in apple polyphenol pharmacokinetics between single subjects were observed. Those could be divided into subgroups according to fast or slow rates of polyphenol metabolism. Some subjects showed no detectable metabolism within the study time frame at all. An increase in the total phenolic content over time did not correlate with an observed, highly elevated antioxidant capacity (AOC) in the blood plasma after apple juice consumption. The determined increase of the AOC was rather a result of a high fructose content of the apple juice. No differences in renal excretion were detected between female and male subjects. However, relative concentrations were slightly higher in male subjects. CONCLUSIONS: Apple derived polyphenols can be readily detected in human blood and urine after juice consumption. The existence of sub-populations with different pharmacokinetics suggests significant variations in the individual metabolism rates of polyphenolic substances with implications on bioavailability and potential health effects within the body. TRIAL REGISTRATION: O2413 (Ethics Commissions of Upper Austria) and 415-EP/73/233-2013 Salzburg (Ethics Commissions of Salzburg).

Regular consumption of an antioxidant-rich juice improves oxidative status and causes metabolome changes in healthy adults.

An improvement in oxidative status is associated with a reduction in the incidence of several chronic diseases. However, daily intake of antioxidants in Western diets is decreasing. This study evaluates the effect of daily consumption of an antioxidant-rich juice (ARJ) on oxidative status, cardiovascular disease risk parameters, and untargeted plasma and urine metabolomes. Twenty-eight healthy young adults participated in an 8-week clinical trial by drinking 200 mL of ARJ (pomegranate and grape) daily. At the end of the study, the subjects showed a significant decrease (-29%) in plasma lipid oxidation (malondialdehyde concentration), and a significant increase (+115%) in plasma antioxidant capacity. Plasma and urine metabolomes were also significantly modified and some ions modified in urine were identified, including metabolites of polyphenols, ascorbic acid and biliary acids. No significant changes were observed in lipid profile, inflammation, blood pressure or glycaemia. These results show that incorporating antioxidant-rich beverages into common diets may improve oxidative status in healthy subjects.

Improved high sensitivity analysis of polyphenols and their metabolites by nano-liquid chromatography-mass spectrometry.

This study was conducted to assess the value of a high resolution, high mass accuracy time-of-flight analyzer in combination with nanoliquid chromatography for the analysis of polyphenols and their metabolites. The goal was to create a method that utilizes small volumes of biological fluids and provides a significant improvement in sensitivity compared with existing methods. Accordingly, nanoLC-MS and nanoLC-pseudo-multiple reaction monitoring (MRM) methods were developed that had a lower limit of quantification of 0.5 nM for several polyphenols and were linear over 2-3 orders of magnitude (R(2)>0.999). Using urine samples, the ability to observe and quantify polyphenols in such a complex biological fluid depended on much narrower mass windows (0.050 amu or less) on a TOF analyzer than those used on a quadrupole analyzer (0.7 amu). Although a greater selectivity was possible with the low mass resolution of a triple quadrupole instrument using the MRM approach, for the daidzein metabolite O-DMA, a chromatographically resolvable second peak could only be substantially reduced by using a 0.01 amu mass window. The advantage of a TOF analyzer for product ion data is that the whole MSMS spectrum is collected at high mass accuracy and MRM experiments are conducted in silico after the analysis.

Bioavailability of lemon verbena (Aloysia triphylla) polyphenols in rats: impact of colonic inflammation.

Lemon verbena (Aloysia triphylla) infusion, a widely consumed herbal tea, contains significant amounts of polyphenols such as flavone diglucuronides and phenylpropanoid glycosides (mainly verbascoside). We have recently shown that lemon verbena infusion offers beneficial effects against dextran sodium sulphate (DSS)-induced colonic inflammation in rats. The present study aimed to evaluate the bioavailability and intestinal absorption of polyphenols derived from lemon verbena infusion in both healthy and colitic rats. For this purpose, lemon verbena infusion was given to rats ad libitum for 14 d, and then 4 % DSS was added to the infusion for 7 d. Before and after DSS administration, 24 h urinary excretion of polyphenols was determined. Flavones were excreted in the urine as conjugated aglycones, and their excretion was not significantly altered by colonic inflammation. Only trace amounts of verbascoside were excreted in the urine, but various metabolites (hydroxycinnamic acids) were detected. The urinary excretion of hydroxycinnamic acids, particularly that of caffeic acid, increased after DSS administration (P< 0·05). Only flavone aglycones (luteolin and diosmetin) were excreted in the faeces in small proportions (3·2 % of ingested flavones). Intestinal absorption of lemon verbena polyphenols was examined using an in situ intestinal perfusion model. Intestinal absorption of verbascoside and flavone diglucuronides did not significantly differ between the healthy and colitic rats. Collectively, these results show that intestinal absorption and urinary excretion of lemon verbena flavone diglucuronides were not altered by colonic inflammation, but that urinary excretion of hydroxycinnamic acids derived from verbascoside was affected in a colitic situation.

Effects of regular consumption of vitamin C-rich or polyphenol-rich apple juice on cardiometabolic markers in healthy adults: a randomized crossover trial.

PURPOSE: The aim of the present study was to investigate the effects of the consumption of two cloudy apple juices with different polyphenol and vitamin C contents on antioxidant status, cardiometabolic and inflammation markers in healthy young adults. METHODS: Twenty subjects, aged 21-29 years, completed a randomized crossover study. At each 4-week intervention period, the volunteers randomly consumed two glasses (2 × 250 mL/day) of either a vitamin C-rich apple juice (VCR) (60 mg/L vitamin C and 510 mg catechin equivalent/L) or a polyphenol-rich (PR) juice (22 mg/L vitamin C and 993 mg catechin equivalent/L). Blood and urine samples were collected throughout the study, and markers of antioxidant status, glucose metabolism, lipid profile and inflammation were measured. RESULTS: The comparison of the post-intervention minus pre-intervention change revealed differential results in HOMA index, total cholesterol, ICAM-1 and VCAM-1 (P < 0.05) across juices. During the VCR period, plasma antioxidant activity (FRAP) increased (P = 0.031), while ICAM-1 and total cholesterol showed a trend to decrease (P = 0.060 and P = 0.094, respectively). During the PR period, plasma insulin and HOMA increased, and total glutathione decreased (P < 0.05). CONCLUSIONS: A joint consumption of apple juice natural antioxidants such as vitamin C and polyphenols might provide mild favorable effects on cardiometabolic markers, as compared to apple polyphenols alone.