Biofortified Yellow-Fleshed Potatoes Provide More Absorbable Zinc than a Commonly Consumed Variety: A Randomized Trial Using Stable Isotopes in Women in the Peruvian Highlands.

BACKGROUND: Zinc-biofortified potatoes have considerable potential to reduce zinc deficiency because of their low levels of phytate, an inhibitor of zinc absorption, and their high consumption, especially in the Andean region of Peru. OBJECTIVES: The purpose of this study was to measure fractional and total zinc absorption from a test meal of biofortified compared with regular potatoes. METHODS: We undertook a single-blinded randomized crossover study (using 67Zn and 70Zn stable isotopes) in which 37 women consumed 500-g biofortified or regular potatoes twice a day. Urine samples were collected to determine fractional and total zinc absorption. RESULTS: The zinc content of the biofortified potato and regular potato was 0.48 (standard deviation [SD]: 0.02) and 0.32 (SD: 0.03) mg/100 g fresh weight, respectively. Mean fractional zinc absorption (FZA) from the biofortified potatoes was lower than from the regular potatoes, 20.8% (SD: 5.4%) and 25.5% (SD: 7.0%), respectively (P < 0.01). However, total zinc absorbed was significantly higher (0.49; SD: 0.13 and 0.40; SD: 0.11 mg/500 g, P < 0.01, respectively). CONCLUSIONS: The results of this study demonstrate that biofortified potatoes provide more absorbable zinc than regular potatoes. Zinc-biofortified potatoes could contribute toward reducing zinc deficiency in populations where potatoes are a staple food. This trial was registered at clinicaltrials.gov as NCT05154500.

Total Iron Absorbed from Iron-Biofortified Potatoes Is Higher than that from Nonbiofortified Potatoes: A Randomized Trial Using Stable Iron Isotopes in Women from the Peruvian Highlands.

BACKGROUND: Yellow-fleshed potatoes biofortified with iron have been developed through conventional breeding, but the bioavailability of iron is unknown. OBJECTIVES: Our objective was to measure iron absorption from an iron-biofortified yellow-fleshed potato clone in comparison with a nonbiofortified yellow-fleshed potato variety. METHODS: We conducted a single-blinded, randomized, crossover, multiple-meal intervention study. Women (n = 28; mean ± SD plasma ferritin 21.3 ± 3.3 µg/L) consumed 10 meals (460 g) of both potatoes, each meal extrinsically labeled with either 58Fe sulfate (biofortified) or 57Fe sulfate (nonfortified), on consecutive days. Iron absorption was estimated from iron isotopic composition in erythrocytes 14 d after administration of the final meal. RESULTS: Mean ± SD iron, phytic acid, and ascorbic acid concentrations in iron-biofortified and the nonfortified potato meals (mg/per 100 mg) were 0.63 ± 0.01 and 0.31 ± 0.01, 39.34 ± 3.04 and 3.10 ± 1.72, and 7.65 ± 0.34 and 3.74 ± 0.39, respectively (P < 0.01), whereas chlorogenic acid concentrations were 15.14 ± 1.72 and 22.52 ± 3.98, respectively (P < 0.05). Geometric mean (95% CI) fractional iron absorption from the iron-biofortified clone and the nonbiofortified variety were 12.1% (10.3%-14.2%) and 16.6% (14.0%-19.6%), respectively (P < 0.001). Total iron absorption from the iron-biofortified clone and the nonbiofortified variety were 0.35 mg (0.30-0.41 mg) and 0.24 mg (0.20-0.28 mg) per 460 g meal, respectively (P < 0.001). CONCLUSIONS: TIA from iron-biofortified potato meals was 45.8% higher than that from nonbiofortified potato meals, suggesting that iron biofortification of potatoes through conventional breeding is a promising approach to improve iron intake in iron-deficient women. The study was registered at www. CLINICALTRIALS: gov as Identifier number NCT05154500.

Microbiomes in physiology: insights into 21st-century global medical challenges.

NEW FINDINGS: What is the topic of this review? The role of the gut microbiome in physiology and how it can be targeted as an effective strategy against two of the most important global medical challenges of our time, namely, metabolic diseases and antibacterial resistance. What advances does it highlight? The critical roles of the microbiome in regulating host physiology and how microbiome analysis is useful for disease stratification to enable informed clinical decisions and develop interventions such as faecal microbiota transplantation, prebiotics and probiotics. Also, the limitations of microbiome modulation, including the potential for probiotics to enhance antimicrobial resistance gene reservoirs, and that currently a 'healthy microbiome' that can be used as a biobank for transplantation is yet to be defined. ABSTRACT: The human gut microbiome is a key factor in the development of metabolic diseases and antimicrobial resistance, which are among the greatest global medical challenges of the 21st century. A recent symposium aimed to highlight state-of-the-art evidence for the role of the gut microbiome in physiology, from childhood to adulthood, and the impact this has on global disease outcomes, ageing and antimicrobial resistance. Although the gut microbiome is established early in life, over time the microbiome and its components including metabolites can become perturbed due to changes such as dietary habits, use of antibiotics and age. As gut microbial metabolites, including short-chain fatty acids, secondary bile acids and trimethylamine-N-oxide, can interact with host receptors including G protein-coupled receptors and can alter host metabolic fluxes, they can significantly affect physiological homoeostasis leading to metabolic diseases. These metabolites can be used to stratify disease phenotypes such as irritable bowel syndrome and adverse events after heart failure and allow informed decisions on clinical management and treatment. While strategies such as use of probiotics, prebiotics and faecal microbiota transplantation have been proposed as interventions to treat and prevent metabolic diseases and antimicrobial resistance, caution must be exercised, first due to the potential of probiotics to enhance antimicrobial resistance gene reservoirs, and second, a 'healthy gut microbiome' that can be used as a biobank for transplantation is yet to be defined. We highlight that sampling other parts of the gastrointestinal tract may produce more representative data than the faecal microbiome alone.

Activity of antibacterial phytochemicals and their potential use as natural food preservatives.

The risk to human health from bacterial foodborne infection is presently controlled by the addition of antimicrobial preservatives to food. However, the use of chemical preservatives such as sodium nitrite poses a health risk in themselves with concerns around carcinogenic effects. This makes the development of improved preservatives a priority for the food industry.One promising source of novel antimicrobial compounds can be found in nature; phytochemicals, in particular polyphenols are secondary metabolites produced by plants for numerous purposes including antimicrobial defence. There has been significant study of phytochemicals; including quantifying their antimicrobial activity, potential to synergise with current antibiotics and the feasibility of their application as natural food preservatives. However, there remains significant uncertainty about the relative antimicrobial efficacy of different phytochemicals, their mechanisms of action (MOA) and the potential for emergence of bacterial resistance to their effects. This review summarizes recent work relevant to the potential development of phytochemicals as antimicrobial agents.

The relationship between urinary polyphenol metabolites and dietary polyphenol intakes in young adults.

Spot urinary polyphenols have potential as a biomarker of polyphenol-rich food intakes. The aim of this study is to explore the relationship between spot urinary polyphenols and polyphenol intakes from polyphenol-rich food sources. Young adults (18-24 years old) were recruited into a sub-study of an online intervention aimed at improving diet quality. Participants' intake of polyphenols and polyphenol-rich foods was assessed at baseline and 3 months using repeated 24-h recalls. A spot urine sample was collected at each session, with samples analysed for polyphenol metabolites using LC-MS. To assess the strength of the relationship between urinary polyphenols and dietary polyphenols, Spearman correlations were used. Linear mixed models further evaluated the relationship between polyphenol intakes and urinary excretion. Total urinary polyphenols and hippuric acid (HA) demonstrated moderate correlation with total polyphenol intakes (rs = 0·29-0·47). HA and caffeic acid were moderately correlated with polyphenols from tea/coffee (rs = 0·26-0·46). Using linear mixed models, increases in intakes of total polyphenols or polyphenols from tea/coffee or oil resulted in a greater excretion of HA, whereas a negative relationship was observed between soya polyphenols and HA, suggesting that participants with higher intakes of soya polyphenols had a lower excretion of HA. Findings suggest that total urinary polyphenols may be a promising biomarker of total polyphenol intakes foods and drinks and that HA may be a biomarker of total polyphenol intakes and polyphenols from tea/coffee. Caffeic acid warrants further investigation as a potential biomarker of polyphenols from tea/coffee.

Higher bacterial DNAemia can affect the impact of a polyphenol-rich dietary pattern on biomarkers of intestinal permeability and cardiovascular risk in older subjects.

PURPOSE: Aging can be characterized by increased systemic low-grade inflammation, altered gut microbiota composition, and increased intestinal permeability (IP). The intake of polyphenol-rich foods is proposed as a promising strategy to positively affect the gut microbiota-immune system-intestinal barrier (IB) axis. In this context, we tested the hypothesis that a PR-dietary intervention would affect the presence of bacterial factors in the bloodstream of older adults. METHODS: We collected blood samples within a randomized, controlled, crossover intervention trial in which older volunteers (n = 51) received a polyphenol-enriched and a control diet. We quantified the presence of bacterial DNA in blood by qPCR targeting the 16S rRNA gene (16S; bacterial DNAemia). Blood DNA was taxonomically profiled via 16S sequencing. RESULTS: Higher blood 16S levels were associated with higher BMI and markers of IP, inflammation, and dyslipidemia. PR-intervention did not significantly change bacterial DNAemia in the older population (P = 0.103). Nonetheless, the beneficial changes caused by the polyphenol-enriched diet were greatest in participants with higher bacterial DNAemia, specifically in markers related to IP, inflammation and dyslipidemia, and in fecal bacterial taxa. Finally, we found that the bacterial DNA detected in blood mostly belonged to γ-Proteobacteria, whose abundance significantly decreased after the polyphenol-rich diet in subjects with higher bacterial DNAemia at baseline. CONCLUSIONS: This study shows that older subjects with higher bacterial DNAemia experienced a beneficial effect from a polyphenol-rich diet. Bacterial DNAemia may be a further relevant marker for the identification of target populations that could benefit more from a protective dietary treatment. REGISTRATION: This trial was retrospectively registered at www.isrctn.org (ISRCTN10214981) on April 28, 2017.

Transcriptional and Post-Translational Regulation of Junctional Adhesion Molecule-B (JAM-B) in Leukocytes under Inflammatory Stimuli.

Junctional adhesion molecules (JAMs; comprising JAM-A, -B and -C) act as receptors for viruses, mediate cell permeability, facilitate leukocyte migration during sterile and non-sterile inflammation and are important for the maintenance of epithelial barrier integrity. As such, they are implicated in the development of both communicable and non-communicable chronic diseases. Here, we investigated the expression and regulation of JAM-B in leukocytes under pathogen- and host-derived inflammatory stimuli using immunoassays, qPCR and pharmacological inhibitors of inflammatory signalling pathways. We show that JAM-B is expressed at both the mRNA and protein level in leukocytes. JAM-B protein is localised to the cytoplasm, Golgi apparatus and in the nucleus around ring-shaped structures. We also provide evidence that JAM-B nuclear localisation occurs via the classical importin-α/ß pathway, which is likely mediated through JAM-B protein nuclear localisation signals (NLS) and export signals (NES). In addition, we provide evidence that under both pathogen- and host-derived inflammatory stimuli, JAM-B transcription is regulated via the NF-κB-dependent pathways, whereas at the post-translational level JAM-B is regulated by ubiquitin-proteosome pathways. Anaphase-promoting ubiquitin ligase complex (APC/C) and herpes simplex virus-associated ubiquitin-specific protease (HAUSP/USP) were identified as candidates for JAM-B ubiquitination and de-ubiquitination, respectively. The expression and regulation of JAM-B in leukocytes reported here is a novel observation and contrasts with previous reports. The data reported here suggest that JAM-B expression in leukocytes is under the control of common inflammatory pathways.

The use of an in-vitro batch fermentation (human colon) model for investigating mechanisms of TMA production from choline, L-carnitine and related precursors by the human gut microbiota.

PURPOSE: Plasma trimethylamine-N-oxide (TMAO) levels have been shown to correlate with increased risk of metabolic diseases including cardiovascular diseases. TMAO exposure predominantly occurs as a consequence of gut microbiota-dependent trimethylamine (TMA) production from dietary substrates including choline, carnitine and betaine, which is then converted to TMAO in the liver. Reducing microbial TMA production is likely to be the most effective and sustainable approach to overcoming TMAO burden in humans. Current models for studying microbial TMA production have numerous weaknesses including the cost and length of human studies, differences in TMA(O) metabolism in animal models and the risk of failing to replicate multi-enzyme/multi-strain pathways when using isolated bacterial strains. The purpose of this research was to investigate TMA production from dietary precursors in an in-vitro model of the human colon. METHODS: TMA production from choline, L-carnitine, betaine and γ-butyrobetaine was studied over 24-48 h using an in-vitro human colon model with metabolite quantification performed using LC-MS. RESULTS: Choline was metabolised via the direct choline TMA-lyase route but not the indirect choline-betaine-TMA route, conversion of L-carnitine to TMA was slower than that of choline and involves the formation of the intermediate γ-BB, whereas the Rieske-type monooxygenase/reductase pathway for L-carnitine metabolism to TMA was negligible. The rate of TMA production from precursors was choline > carnitine > betaine > γ-BB. 3,3-Dimethyl-1-butanol (DMB) had no effect on the conversion of choline to TMA. CONCLUSION: The metabolic routes for microbial TMA production in the colon model are consistent with observations from human studies. Thus, this model is suitable for studying gut microbiota metabolism of TMA and for screening potential therapeutic targets that aim to attenuate TMA production by the gut microbiota. TRIAL REGISTRATION NUMBER: NCT02653001 ( http://www.clinicaltrials.gov ), registered 12 Jan 2016.

Development and Validation of a LC-MS/MS Technique for the Analysis of Short Chain Fatty Acids in Tissues and Biological Fluids without Derivatisation Using Isotope Labelled Internal Standards.

The gut microbiota is critical to the maintenance of physiological homeostasis and as such is implicated in a range of diseases such as colon cancer, ulcerative colitis, diabetes, cardiovascular diseases, and neurodegenerative diseases. Short chain fatty acids (SCFAs) are key metabolites produced by the gut microbiota from the fermentation of dietary fibre. Here we present a novel, sensitive, and direct LC-MS/MS technique using isotopically labelled internal standards without derivatisation for the analysis of SCFAs in different biological matrices. The technique has significant advantages over the current widely used techniques based on sample derivatization and GC-MS analysis, including fast and simple sample preparation and short LC runtime (10 min). The technique is specific and sensitive for the quantification of acetate, butyrate, isobutyrate, isovalerate, lactate, propionate and valerate. The limits of detection were all 0.001 mM except for acetate which was 0.003 mM. The calibration curves for all the analytes were linear with correlation coefficients r2 > 0.998. The intra- and inter-day precisions in three levels of known concentrations were <12% and <20%, respectively. The quantification accuracy ranged from 92% to 120%. The technique reported here offers a valuable analytical tool for use in studies of SCFA production in the gut and their distribution to host tissues.

Polyphenols bind to low density lipoprotein at biologically relevant concentrations that are protective for heart disease.

There is ample evidence in the epidemiological literature that polyphenols, the major non-vitamin antioxidants in plant foods and beverages, have a beneficial effect on heart disease. Until recently other mechanisms which polyphenols exhibit such as cell signaling and regulating nitric oxide bioavailability have been investigated. The oxidation theory of atherosclerosis implicates LDL oxidation as the beginning step in this process. Nine polyphenols from eight different classes and several of their O-methylether, O-glucuronide and O-sulfate metabolites have been shown in this study to bind to the lipoproteins and protect them from oxidation at lysosomal/inflammatory pH (5.2), and physiological pH (7.4). Polyphenols bind to the apoprotein at pH 7.4 with Kb > 106 M-1 and the number of molecules of polyphenols bound per LDL particle under saturation conditions varied from 0.4 for ferulic acid to 13.1 for quercetin. Competition studies between serum albumin and LDL show that substantial lipoprotein binding occurs even in the presence of a great molar excess of albumin, the major blood protein. These in vitro results are borne out by published human supplementation studies showing that polyphenol metabolites from red wine, olive oil and coffee are found in LDL even after an overnight fast. A single human supplementation with various fruit juices, coffee and tea also produced an ex vivo protection against lipoprotein oxidation under postprandial conditions. This in vivo binding is heart-protective based on published olive oil consumption studies. Relevant to heart disease, we hypothesize that the binding of polyphenols and metabolites to LDL functions as a transport mechanism to carry these antioxidants to the arterial intima, and into endothelial cells and macrophages. Extracellular and intracellular polyphenols and their metabolites are heart-protective by many mechanisms and can also function as potent "intraparticle" and intracellular antioxidants due to their localized concentrations that can reach as high as the micromolar level. Low plasma concentrations make polyphenols and their metabolites poor plasma antioxidants but their concentration in particles such as lipoproteins and cells is high enough for polyphenols to provide cardiovascular protection by direct antioxidant effects and by other mechanisms such as cell signaling.

Effect of a polyphenol-rich dietary pattern on intestinal permeability and gut and blood microbiomics in older subjects: study protocol of the MaPLE randomised controlled trial.

BACKGROUND: During aging, alterations of the intestinal microbial ecosystem can occur contributing to immunosenescence, inflamm-aging and impairment of intestinal barrier function (increased intestinal permeability; IP). In the context of a diet-microbiota-IP axis in older subjects, food bioactives such as polyphenols may play a beneficial modulatory role. METHODS: MaPLE is a project centered on a randomized, controlled cross-over dietary intervention trial [polyphenol-rich diet (PR-diet) versus control diet (C-diet)] targeted to older people (≥ 60 y) living in a well-controlled setting (i.e. nursing home). The 8-week interventions are separated by an 8-week wash-out period. Three small portions per day of selected polyphenol-rich foods are consumed during intervention in substitution of other comparable products within the C-diet. Biological samples are collected before and after each treatment period to evaluate markers related to IP, inflammation, vascular function, oxidative stress, gut and blood microbiomics, metabolomics. A sample size of 50 subjects was defined based on IP as primary outcome. DISCUSSION: Evidence that increasing the consumption of polyphenol-rich food products can positively affect intestinal microbial ecosystem resulting in reduced IP and decreased translocation of inflammogenic bacterial factors into the bloodstream will be provided. The integration of data from gut and blood microbiomics, metabolomics and other IP-related markers will improve the understanding of the beneficial effect of the intervention in the context of polyphenols-microbiota-IP interactions. Finally, findings obtained will provide a proof of concept of the reliability of the dietary intervention, also contributing to future implementations of dietary guidelines directed to IP management in the older and other at risk subjects. TRIAL REGISTRATION: The trial is registered at (ISRCTN10214981); April 28, 2017.

Effect of an egg ovalbumin-derived protein hydrolysate on blood pressure and cardiovascular risk in adults with a mildly elevated blood pressure: a randomized placebo-controlled crossover trial.

PURPOSE: While animal and in vitro data demonstrate vasodilatory effects of egg white-derived peptides, human studies are lacking. We investigated for the first time the effects of an egg ovalbumin-derived protein hydrolysate on blood pressure (BP) and cardiovascular risk. METHODS: A double-blind, placebo-controlled randomized crossover trial was implemented in 75 adults aged 50-70 years with systolic BP (130-≤ 150 mmHg). Participants were randomized to an egg ovalbumin-derived protein hydrolysate (3 g/day) or placebo (3 g/day). Participants completed two 6-week periods separated by a 3-week washout. RESULTS: Data from 65 participants with a mean systolic BP (135.1 ± 11 mmHg) were included. Mean office and central BP and arterial stiffness (assessed by carotid-femoral pulse wave velocity (cfPWV) or pulse wave analysis (PWA)) did not change over time and no significant differences were observed between the egg protein hydrolysate and placebo groups (P > 0.05). Similarly, no significant effects of this egg ovalbumin-derived protein hydrolysate on blood lipid and glucose concentrations (P > 0.05) were observed. CONCLUSION: This is the first dietary intervention to investigate the effects of egg ovalbumin-derived protein hydrolysates on cardiovascular risk in humans. Despite promising findings from animal and in vitro studies, this RCT does not support the hypothesis that consumption of an egg ovalbumin-derived protein hydrolysate for 6 weeks in adults with a high-normal BP results in a reduction in BP or the modification of cardiovascular risk.

Targeting the delivery of dietary plant bioactives to those who would benefit most: from science to practical applications.

BACKGROUND: A healthy diet and optimal lifestyle choices are amongst the most important actions for the prevention of cardiometabolic diseases. Despite this, it appears difficult to convince consumers to select more nutritious foods. Furthermore, the development and production of healthier foods do not always lead to economic profits for the agro-food sector. Most dietary recommendations for the general population represent a "one-size-fits-all approach" which does not necessarily ensure that everyone has adequate exposure to health-promoting constituents of foods. Indeed, we now know that individuals show a high variability in responses when exposed to specific nutrients, foods, or diets. PURPOSE: This review aims to highlight our current understanding of inter-individual variability in response to dietary bioactives, based on the integration of findings of the COST Action POSITIVe. We also evaluate opportunities for translation of scientific knowledge on inter-individual variability in response to dietary bioactives, once it becomes available, into practical applications for stakeholders, such as the agro-food industry. The potential impact from such applications will form an important impetus for the food industry to develop and market new high quality and healthy foods for specific groups of consumers in the future. This may contribute to a decrease in the burden of diet-related chronic diseases.

Inhibitory Effects of Quercetin and Its Human and Microbial Metabolites on Xanthine Oxidase Enzyme.

Quercetin is an abundant flavonoid in nature and is used in several dietary supplements. Although quercetin is extensively metabolized by human enzymes and the colonic microflora, we have only few data regarding the pharmacokinetic interactions of its metabolites. Therefore, we investigated the interaction of human and microbial metabolites of quercetin with the xanthine oxidase enzyme. Inhibitory effects of five conjugates and 23 microbial metabolites were examined with 6-mercaptopurine and xanthine substrates (both at 5 µM), employing allopurinol as a positive control. Quercetin-3'-sulfate, isorhamnetin, tamarixetin, and pyrogallol proved to be strong inhibitors of xanthine oxidase. Sulfate and methyl conjugates were similarly strong inhibitors of both 6-mercaptopurine and xanthine oxidations (IC50 = 0.2-0.7 µM); however, pyrogallol inhibited xanthine oxidation (IC50 = 1.8 µM) with higher potency vs. 6-MP oxidation (IC50 = 10.1 µM). Sulfate and methyl conjugates were approximately ten-fold stronger inhibitors (IC50 = 0.2-0.6 µM) of 6-mercaptopurine oxidation than allopurinol (IC50 = 7.0 µM), and induced more potent inhibition compared to quercetin (IC50 = 1.4 µM). These observations highlight that some quercetin metabolites can exert similar or even a stronger inhibitory effect on xanthine oxidase than the parent compound, which may lead to the development of quercetin-drug interactions (e.g., with 6-mercaptopurin or azathioprine).

Hydrophobic Interactions Drive Binding between Vascular Endothelial Growth Factor-A (VEGFA) and Polyphenolic Inhibitors.

Some polyphenols have been shown to inhibit, at physiological levels, the VEGF-induced VEGF receptor-2 signaling that causes angiogenesis, allegedly by direct interaction with VEGF and reducing the binding to its receptor VEGFR2. Surface plasmon resonance was used to measure the parameters of binding between VEGF and polyphenols as well as the nature of the interactions by assessing the effect of physico-chemical changes in the solution. CD spectrometry was used to determine any change in the secondary structure of the protein upon binding. The kinetic parameters (ka, kd, and KD) that characterise the binding to VEGF were measured for both inhibitor and non-inhibitor polyphenolic molecules. The effect of changes in the physico-chemical conditions of the solution where the binding occurred indicated that the nature of the interactions between VEGF and EGCG was predominantly of a hydrophobic nature. CD studies suggested that a change in the secondary structure of the protein occurred upon binding. Direct interaction and binding between VEGF and polyphenol molecules acting as inhibitors of the signaling of VEGFR2 has been measured for the first time. The binding between VEGF and EGCG seemed to be based on hydrophobic interactions and caused a change in the secondary structure of the protein.

4-Week consumption of anthocyanin-rich blood orange juice does not affect LDL-cholesterol or other biomarkers of CVD risk and glycaemia compared with standard orange juice: a randomised controlled trial.

Elevated circulating cholesterol levels are a risk factor for CVD which is also associated with sub-optimal vascular function. There is emerging evidence that anthocyanins can cause beneficial cardio-protective effects by favourably modulating lipoprotein profiles. We compared the effects of blood orange juice which is rich in anthocyanins and blonde orange juice without anthocyanins on LDL-cholesterol and other biomarkers of CVD risk, vascular function and glycaemia. In all, forty-one participants (aged 25-84 years) with a waist circumference >94 cm (men) and >80 cm (women) completed a randomised, open label, two-arm cross-over trial. For 28 d participants ingested (i) 500 ml blood orange juice providing 50 mg anthocyanins/d and (ii) 500 ml blonde orange juice without anthocyanins. There was a minimum 3-week washout period between treatments. LDL-cholesterol and other biomarkers associated with CVD risk and glycaemia were assessed at the start and end of each treatment period. No significant differences were observed in total, HDL- and LDL-cholesterol, TAG, glucose, fructosamine, nitric oxide, C-reactive protein, aortic systolic blood pressure and diastolic blood pressure or carotid-femoral and brachial-ankle pulse wave velocity after 28 d ingestion of blood orange juice compared with standard orange juice. The lack of effect on LDL-cholesterol may be due to the modest concentration of anthocyanins in the blood orange juice.

A One-Step Extraction and Luminescence Assay for Quantifying Glucose and ATP Levels in Cultured HepG2 Cells.

A fluorescence-based enzymatic microplate intracellular glucose assay was designed and fully validated. The method was tested in a hepatocellular cancer cell line (HepG2). Our novel one-step extraction reagent gave stable cell lysates for glucose, adenosine triphosphate (ATP), and total protein determination from the same sample. Limit of detection for glucose was 0.13 µM (26 pmol/well), which is superior to commercially available glucose assays. Both intra- and interday assay imprecision in HepG2 cultures were less than 12% coefficient of variance (CV). In cell lysates spiked with glucose, recovery at two levels varied between 83.70% and 91.81%, and both linearity and stability were acceptable. HepG2 cells treated with agents affecting glucose uptake/metabolism (phloretin, quercetin, quercetin-3'-sulfate, NaF, 3-bromopyruvate, NaN3, oligomycin A, ochratoxin A, cytochalasin B, and anti-GLUT1 antibody) showed dose-dependent changes in glucose and ATP levels without total protein (cell) loss. Finally, we performed flow cytometric glucose uptake measurement in the treated cells using 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose fluorescent glucose analog. Glucose uptake did not always mirror the intracellular glucose levels, which most likely reflects the differences between the two methodologies. However, interpreting data obtained by both methods and taking ATP/protein levels at the same time, one can get information on the mode of action of the compounds.

Meta-Analysis of the Effects of Foods and Derived Products Containing Ellagitannins and Anthocyanins on Cardiometabolic Biomarkers: Analysis of Factors Influencing Variability of the Individual Responses.

Understanding interindividual variability in response to dietary polyphenols remains essential to elucidate their effects on cardiometabolic disease development. A meta-analysis of 128 randomized clinical trials was conducted to investigate the effects of berries and red grapes/wine as sources of anthocyanins and of nuts and pomegranate as sources of ellagitannins on a range of cardiometabolic risk biomarkers. The potential influence of various demographic and lifestyle factors on the variability in the response to these products were explored. Both anthocyanin- and ellagitannin-containing products reduced total-cholesterol with nuts and berries yielding more significant effects than pomegranate and grapes. Blood pressure was significantly reduced by the two main sources of anthocyanins, berries and red grapes/wine, whereas waist circumference, LDL-cholesterol, triglycerides, and glucose were most significantly lowered by the ellagitannin-products, particularly nuts. Additionally, we found an indication of a small increase in HDL-cholesterol most significant with nuts and, in flow-mediated dilation by nuts and berries. Most of these effects were detected in obese/overweight people but we found limited or non-evidence in normoweight individuals or of the influence of sex or smoking status. The effects of other factors, i.e., habitual diet, health status or country where the study was conducted, were inconsistent and require further investigation.

Acute Consumption of Flavan-3-ol-Enriched Dark Chocolate Affects Human Endogenous Metabolism.

Flavan-3-ols and methylxanthines have potential beneficial effects on human health including reducing cardiovascular risk. We performed a randomized controlled crossover intervention trial to assess the acute effects of consumption of flavan-3-ol-enriched dark chocolate, compared with standard dark chocolate and white chocolate, on the human metabolome. We assessed the metabolome in urine and blood plasma samples collected before and at 2 and 6 h after consumption of chocolates in 42 healthy volunteers using a nontargeted metabolomics approach. Plasma samples were assessed and showed differentiation between time points with no further separation among the three chocolate treatments. Multivariate statistics applied to urine samples could readily separate the postprandial time points and distinguish between the treatments. Most of the markers responsible for the multivariate discrimination between the chocolates were of dietary origin. Interestingly, small but significant level changes were also observed for a subset of endogenous metabolites. 1H NMR revealed that flavan-3-ol-enriched dark chocolate and standard dark chocolate reduced urinary levels of creatinine, lactate, some amino acids, and related degradation products and increased the levels of pyruvate and 4-hydroxyphenylacetate, a phenolic compound of bacterial origin. This study demonstrates that an acute chocolate intervention can significantly affect human metabolism.

Design, formulation and sensory evaluation of a polyphenol-rich food placebo: an example of aronia juice for food intervention studies.

Products suitable for use as controls in food interventions designed to demonstrate the role of minor components are largely lacking. In the present study, we aimed to develop a formulation to be used as a placebo in a clinical trial designed to assess the effects of aronia juice polyphenols on platelet function. Three formulations with the same nutrient composition as aronia juice were prepared by mixing various nutrients, artificial colours and flavours with water. The similarity of formulations to aronia juice in terms of taste, colour, smell and texture was assessed by six food panellists. The final placebo was tested for its impact on platelet function, biochemical and anthropometric parameters in a 4-week long study. No significant changes in platelet function, or in several cardiovascular and safety markers were recorded. Formulation suitable for use as a placebo for dietary intervention studies using aronia juice has been developed and demonstrated to be well tolerated in humans.