Performance of Urinary Phenyl-γ-Valerolactones as Biomarkers of Dietary Flavan-3-ol Exposure.

BACKGROUND: Phenyl-γ-valerolactones (PVLs) have been identified as biomarkers of dietary flavan-3-ol exposure, although their utility requires further characterization. OBJECTIVES: We investigated the performance of a range of PVLs as biomarkers indicative of flavan-3-ol intake. METHODS: We report the results of 2 companion studies: a 5-way randomized crossover trial (RCT) and an observational cross-sectional study. In the RCT (World Health Organization, Universal Trial Number: U1111-1236-7988), 16 healthy participants consumed flavan-3-ol-rich interventions (of apple, cocoa, black tea, green tea, or water [control]) for 1 d each. First morning void samples and 24-h urine samples were collected with diet standardized throughout. For each participant, 1 intervention period was extended (to 2 d) to monitor PVL kinetics after repeat exposure. In the cross-sectional study, 86 healthy participants collected 24-h urine samples, and concurrent weighed food diaries from which flavan-3-ol consumption was estimated using Phenol-Explorer. A panel of 10 urinary PVLs was quantified using liquid chromatography tandem mass spectrometry. RESULTS: In both studies, 2 urinary PVLs [5-(3'-hydroxyphenyl)-γ-valerolactone-4'-sulfate and putatively identified 5-(4'-hydroxyphenyl)-γ-valerolactone-3'-glucuronide] were the principal compounds excreted (>75%). In the RCT, the sum of these PVLs was significantly higher than the water (control) after each intervention; individually, there was a shift from sulfation toward glucuronidation as the total excretion of PVLs increased across the different interventions. In the extended RCT intervention period, no accumulation of these PVLs was observed after consecutive days of treatment, and after withdrawal of treatment on the third day, there was a return toward negligible PVL excretion. All results were consistent, whether compounds were measured in 24-h urine or first morning void samples. In the observational study, the sum of the principal PVLs correlated dose dependently (Rs = 0.37; P = 0.0004) with dietary flavan-3-ol intake, with similar associations for each individually. CONCLUSIONS: Urinary 5-(3'-hydroxyphenyl)-γ-valerolactone-4'-sulfate and putatively identified 5-(4'-hydroxyphenyl)-γ-valerolactone-3'-glucuronide are recommended biomarkers for dietary flavan-3-ol exposure.

Cocoa extract exerts sex-specific anti-diabetic effects in an aggressive type-2 diabetes model: A pilot study.

Type 2 diabetes (T2D) is characterized by hyperglycemia and insulin resistance. Cocoa may slow T2D development and progression. This study employed male and female BTBR.Cg-Lepob/ob/WiscJ (ob/ob) and wild type (WT) controls to assess the potential for cocoa to ameliorate progressive T2D and compare responses between sexes. Mice received diet without (WT, ob/ob) or with cocoa extract (ob/ob + c) for 10 weeks. Acute cocoa reduced fasting hyperglycemia in females, but not males, after 2 weeks. Chronic cocoa supplementation (6-10 weeks) ameliorated hyperinsulinemia in males and worsened hyperlipidemia and hyperinsulinemia in females, yet also preserved and enhanced beta cell survival in females. The underlying mechanisms of these differences warrant further study. If sex differences are apparent in subsequent preclinical studies, clinical studies will be warranted to establish whether these differences are relevant in humans. Sex differences may need to be considered when designing human dietary interventions for T2D.

Microbial Metabolites of Flavanols in Urine are Associated with Enhanced Anti-Proliferative Activity in Bladder Cancer Cells In Vitro.

Flavanols are metabolized by the gut microbiota to bioavailable metabolites, and the absorbed fraction is excreted primarily via urine. Uroepithelial cells are thus a potential site of activity due to exposure to high concentrations of these compounds. Chemoprevention by flavanols may be partly due to these metabolites. In Vitro work in this area relies on a limited pool of commercially available microbial metabolites, and little has been done in bladder cancer. The impact of physiologically relevant mixtures of flavanols and their metabolites remains unknown. Rats were fed various flavanols and urine samples, approximating the bioavailable metabolome, were collected. Urines were profiled by UPLC-MS/MS, and their anti-proliferative activities were assayed In Vitro in four bladder cancer models. Significant interindividual variability was observed for composition and proliferation. Microbial metabolite concentrations (valerolactones, phenylalkyl acids and hippuric acids) were positively associated with reduced bladder cancer proliferation In Vitro, while native flavanols were poorly correlated with activity. These results suggest that microbial metabolites may be responsible for chemoprevention in uroepithelial cells following flavanol consumption. This highlights the potential to use individual genetics and microbial metabotyping to design personalized dietary interventions for cancer prevention and/or adjuvant therapy to reduce bladder cancer incidence and improve outcomes.

Strawberry Consumption, Cardiometabolic Risk Factors, and Vascular Function: A Randomized Controlled Trial in Adults with Moderate Hypercholesterolemia.

BACKGROUND: Certain fruits, such as strawberries, may impart cardiometabolic benefits due to their phytochemical content. OBJECTIVES: Study aims were to assess the effects of strawberry intake on cardiometabolic risk factors and vascular endothelial function in adults with moderate hypercholesterolemia. METHODS: This study was a randomized, controlled, double-blinded, 2-arm, 2-period (4-wk/period) crossover trial. Adults (n = 34; male/female 1:1; mean ± SEM age, 53 ± 1 y; BMI, 31 ± 1 kg/m2;  LDL cholesterol, 133 ± 3 mg/dL) were randomly allocated to 1 of 2 study sequences in a 1:1 ratio. Participants drank study beverages twice daily containing freeze-dried strawberry powder (2 × 25 g) or energy-, volume-matched control powder for 4 wk separated by a 4-wk washout. The primary outcome variable was the difference in fasting LDL cholesterol after 4-wk interventions. Secondary outcomes were metabolic markers, inflammation, quantitative (poly)phenolic metabolomics, flow-mediated dilation (FMD), and blood pressure (BP), with the latter (FMD, BP) also assessed acutely at 1 h and 2 h after a 50-g bolus strawberry or control beverage. Mixed-model analysis of repeated measures via PROC MIXED, PC-SAS was performed on primary and secondary outcome variables. RESULTS: LDL cholesterol did not differ after the 4-wk interventions (P > 0.05), nor did fasting total cholesterol, triglycerides, glucose, insulin, high-sensitivity C-reactive protein, FMD, or BP (all P > 0.05). Significant intervention-by-hour interaction for FMD (P = 0.03) and BP (P = 0.05) revealed increased FMD at 1 h after strawberry compared with control by 1.5 ± 0.38% (P = 0.0008) and attenuated systolic BP at 2 h by 3.1 ± 0.99 mmHg (P = 0.02). Select phenolic metabolites increased significantly (P < 0.05) in blood following strawberry consumption while others decreased, including 3-(4-methoxyphenyl)propanoic acid-3-O-glucuronide, which was significantly correlated with increased FMD (P < 0.05). CONCLUSION: Strawberries may improve vascular health, independent of other metabolic changes. The effect may be related to changes in microbial-derived phenolic metabolites after strawberry consumption influencing endothelial function. Data support inclusion of strawberries in a heart-healthy diet in adults with moderate hypercholesterolemia.This trial was registered at clinicaltrials.gov as NCT02612090.

Contribution of Berry Polyphenols to the Human Metabolome.

Diets rich in berries provide health benefits, however, the contribution of berry phytochemicals to the human metabolome is largely unknown. The present study aimed to establish the impact of berry phytochemicals on the human metabolome. A "systematic review strategy" was utilized to characterize the phytochemical composition of the berries most commonly consumed in the USA; (poly)phenols, primarily anthocyanins, comprised the majority of reported plant secondary metabolites. A reference standard library and tandem mass spectrometry (MS/MS) quantitative metabolomics methodology were developed and applied to serum/plasma samples from a blueberry and a strawberry intervention, revealing a diversity of benzoic, cinnamic, phenylacetic, 3-(phenyl)propanoic and hippuric acids, and benzyldehydes. 3-Phenylpropanoic, 2-hydroxybenzoic, and hippuric acid were highly abundant (mean > 1 µM). Few metabolites at concentrations above 100 nM changed significantly in either intervention. Significant intervention effects (P < 0.05) were observed for plasma/serum 2-hydroxybenzoic acid and hippuric acid in the blueberry intervention, and for 3-methoxyphenylacetic acid and 4-hydroxyphenylacetic acid in the strawberry intervention. However, significant within-group effects for change from baseline were prevalent, suggesting that high inter-individual variability precluded significant treatment effects. Berry consumption in general appears to cause a fluctuation in the pools of small molecule metabolites already present at baseline, rather than the appearance of unique berry-derived metabolites, which likely reflects the ubiquitous nature of (poly)phenols in the background diet.

Cardiovascular Mechanisms of Action of Anthocyanins May Be Associated with the Impact of Microbial Metabolites on Heme Oxygenase-1 in Vascular Smooth Muscle Cells.

Anthocyanins are reported to have cardio-protective effects, although their mechanisms of action remain elusive. We aimed to explore the effects of microbial metabolites common to anthocyanins and other flavonoids on vascular smooth muscle heme oxygenase-1 (HO-1) expression. Thirteen phenolic metabolites identified by previous anthocyanin human feeding studies, as well as 28 unique mixtures of metabolites and their known precursor structures were explored for their activity on HO-1 protein expression in rat aortic smooth muscle cells (RASMCs). No phenolic metabolites were active when treated in isolation; however, five mixtures of phenolic metabolites significantly increased HO-1 protein expression (127.4-116.6%, p ≤ 0.03). The present study demonstrates that phenolic metabolites of anthocyanins differentially affect HO-1 activity, often having additive, synergistic or nullifying effects.

The Bioavailability, Transport, and Bioactivity of Dietary Flavonoids: A Review from a Historical Perspective.

Flavonoids are plant-derived dietary components with a substantial impact on human health. Research has expanded massively since it began in the 1930s, and the complex pathways involved in bioavailability of flavonoids in the human body are now well understood. In recent years, it has been appreciated that the gut microbiome plays a major role in flavonoid action, but much progress still needs to be made in this area. Since the first publications on the health effects of flavonoids, their action is understood to protect against various stresses, but the mechanism of action has evolved from the now debunked simple direct antioxidant hypothesis into an understanding of the complex effects on molecular targets and enzymes in specific cell types. This review traces the development of the field over the past 8 decades, and indicates the current state of the art, and how it was reached. Future recommendations based on this historical analysis are (a) to focus on key areas of flavonoid action, (b) to perform human intervention studies focusing on bioavailability and protective effects, and (c) to carry out cellular in vitro experiments using appropriate cells together with the chemical form of the flavonoid found at the site of action; this could be the native form of compounds found in the food for studies on digestion and the intestine, the conjugated metabolites found in the blood after absorption in the small intestine for studies on cells, or the chemical forms found in the blood and tissues after catabolism by the gut microbiota.

Influence of Ingesting a Flavonoid-Rich Supplement on the Metabolome and Concentration of Urine Phenolics in Overweight/Obese Women.

This study evaluated the effect of ingesting a flavonoid-rich supplement (329 mg/d) on total urine phenolics and shifts in plasma metabolites in overweight/obese female adults using untargeted metabolomics procedures. Participants (N = 103, 18-65 y, BMI ≥ 25 kg/m2) were randomized to flavonoid (F) or placebo (P) groups for 12 weeks with blood and 24 h urine samples collected prestudy and after 4 and 12 weeks in a parallel design. Supplements were prepared as chewable tablets and included vitamin C, wild bilberry fruit extract, green tea leaf extract, quercetin, caffeine, and omega 3 fatty acids. At 4 weeks, urine total phenolics increased 24% in F versus P with similar changes at 12 weeks (interaction effect, P = 0.041). Groups did not differ in markers of inflammation (IL-6, MCP-1, CRP) or oxidative stress (oxLDL, FRAP). Metabolomics data indicated shifts in 63 biochemicals in F versus P with 70% from the lipid and xenobiotics superpathways. The largest fold changes in F were measured for three gut-derived phenolics including 3-methoxycatechol sulfate, 3-(3-hydroxyphenyl)propanoic acid sulfate, and 1,2,3-benzenetriol sulfate (interaction effects, p ≤ 0.050). This randomized clinical trial of overweight/obese women showed that 12 weeks ingestion of a mixed flavonoid nutrient supplement was associated with a corresponding increase in urine total phenolics and gut-derived phenolic metabolites.

Common Phenolic Metabolites of Flavonoids, but Not Their Unmetabolized Precursors, Reduce the Secretion of Vascular Cellular Adhesion Molecules by Human Endothelial Cells.

BACKGROUND: Flavonoids have been implicated in the prevention of cardiovascular disease; however, their mechanisms of action have yet to be elucidated, possibly because most previous in vitro studies have used supraphysiological concentrations of unmetabolized flavonoids, overlooking their more bioavailable phenolic metabolites. OBJECTIVE: We aimed to explore the effects of phenolic metabolites and their precursor flavonoids at physiologically achievable concentrations, in isolation and combination, on soluble vascular cellular adhesion molecule-1 (sVCAM-1). METHOD: Fourteen phenolic acid metabolites and 6 flavonoids were screened at 1 µM for their relative effects on sVCAM-1 secretion by human umbilical vein endothelial cells stimulated with tumor necrosis factor alpha (TNF-α). The active metabolites were further studied for their response at different concentrations (0.01 µM-100 µM), structure-activity relationships, and effect on vascular cellular adhesion molecule (VCAM)-1 mRNA expression. In addition, the additive activity of the metabolites and flavonoids was investigated by screening 25 unique mixtures at cumulative equimolar concentrations of 1 µM. RESULTS: Of the 20 compounds screened at 1 µM, inhibition of sVCAM-1 secretion was elicited by 4 phenolic metabolites, of which protocatechuic acid (PCA) was the most active (-17.2%, P = 0.05). Investigations into their responses at different concentrations showed that PCA significantly reduced sVCAM-1 15.2-36.5% between 1 and 100 µM, protocatechuic acid-3-sulfate and isovanillic acid reduced sVCAM-1 levels 12.2-54.7% between 10 and 100 µM, and protocatechuic acid-4-sulfate and isovanillic acid-3-glucuronide reduced sVCAM-1 secretion 27.6% and 42.8%, respectively, only at 100 µM. PCA demonstrated the strongest protein response and was therefore explored for its effect on VCAM-1 mRNA, where 78.4% inhibition was observed only after treatment with 100 µM PCA. Mixtures of the metabolites showed no activity toward sVCAM-1, suggesting no additive activity at 1 µM. CONCLUSIONS: The present findings suggest that metabolism of flavonoids increases their vascular efficacy, resulting in a diversity of structures of varying bioactivity in human endothelial cells.

Methods for isolating, identifying, and quantifying anthocyanin metabolites in clinical samples.

The metabolic fate of anthocyanins until recently was relatively unknown, primarily as a result of their instability at physiological pH and a lack of published methods for isolating and identifying their metabolites from biological samples. The aim of the present work was to establish methods for the extraction and quantification of anthocyanin metabolites present in urine, serum, and fecal samples. 35 commercial and 10 synthetic analytes, including both known and predicted human and microbial metabolites of anthocyanins, were obtained as reference standards. HPLC and MS/MS conditions were optimized for organic modifier, ionic modifier, mobile phase gradient, flow rate, column type, MS source, and compound dependent parameters. The impact of sorbent, solvent, acid, preservative, elution, and evaporation on solid phase extraction (SPE) efficiency was also explored. The HPLC-MS/MS method validation demonstrated acceptable linearity (R(2), 0.997 ± 0.002) and sensitivity (limits of detection (LODs): urine, 100 ± 375 nM; serum, 104 ± 358 nM; feces 138 ± 344 nM), and the final SPE methods provided recoveries of 88.3 ± 17.8% for urine, 86.5 ± 11.1% for serum, and 80.6 ± 20.9% for feces. The final methods were applied to clinical samples derived from an anthocyanin intervention study, where 36 of the 45 modeled metabolites were detected within urine, plasma, or fecal samples. The described methods provide suitable versatility for the identification and quantification of an extensive series of anthocyanin metabolites for use in future clinical studies exploring absorption, distribution, metabolism, and elimination.

A moderate-fat diet containing pistachios improves emerging markers of cardiometabolic syndrome in healthy adults with elevated LDL levels.

A randomised, cross-over, controlled-feeding study was conducted to evaluate the cholesterol-lowering effects of diets containing pistachios as a strategy for increasing total fat (TF) levels v. a control (step I) lower-fat diet. Ex vivo techniques were used to evaluate the effects of pistachio consumption on lipoprotein subclasses and functionality in individuals (n 28) with elevated LDL levels ( ≥ 2·86 mmol/l). The following test diets (SFA approximately 8 % and cholesterol < 300 mg/d) were used: a control diet (25 % TF); a diet comprising one serving of pistachios per d (1PD; 30 % TF); a diet comprising two servings of pistachios per d (2PD; 34 % TF). A significant decrease in small and dense LDL (sdLDL) levels was observed following the 2PD dietary treatment v. the 1PD dietary treatment (P= 0·03) and following the 2PD dietary treatment v. the control treatment (P= 0·001). Furthermore, reductions in sdLDL levels were correlated with reductions in TAG levels (r 0·424, P= 0·025) following the 2PD dietary treatment v. the control treatment. In addition, inclusion of pistachios increased the levels of functional α-1 (P= 0·073) and α-2 (P= 0·056) HDL particles. However, ATP-binding cassette transporter A1-mediated serum cholesterol efflux capacity (P= 0·016) and global serum cholesterol efflux capacity (P= 0·076) were only improved following the 2PD dietary treatment v. the 1PD dietary treatment when baseline C-reactive protein status was low ( < 103µg/l). Moreover, a significant decrease in the TAG:HDL ratio was observed following the 2PD dietary treatment v. the control treatment (P= 0·036). There was a significant increase in ß-sitosterol levels (P< 0·0001) with the inclusion of pistachios, confirming adherence to the study protocol. In conclusion, the inclusion of pistachios in a moderate-fat diet favourably affects the cardiometabolic profile in individuals with an increased risk of CVD.

Sulforaphane represses matrix-degrading proteases and protects cartilage from destruction in vitro and in vivo.

OBJECTIVE: Sulforaphane (SFN) has been reported to regulate signaling pathways relevant to chronic diseases. The aim of this study was to investigate the impact of SFN treatment on signaling pathways in chondrocytes and to determine whether sulforaphane could block cartilage destruction in osteoarthritis. METHODS: Gene expression, histone acetylation, and signaling of the transcription factors NF-E2-related factor 2 (Nrf2) and NF-κB were examined in vitro. The bovine nasal cartilage explant model and the destabilization of the medial meniscus (DMM) model of osteoarthritis in the mouse were used to assess chondroprotection at the tissue and whole-animal levels. RESULTS: SFN inhibited cytokine-induced metalloproteinase expression in primary human articular chondrocytes and in fibroblast-like synovial cells. SFN acted independently of Nrf2 and histone deacetylase activity to regulate metalloproteinase expression in human articular chondrocytes but did mediate prolonged activation of JNK and p38 MAPK. SFN attenuated NF-κB signaling at least through inhibition of DNA binding in human articular chondrocytes, with decreased expression of several NF-κB-dependent genes. Compared with cytokines alone, SFN (10 µM) abrogated cytokine-induced destruction of bovine nasal cartilage at both the proteoglycan and collagen breakdown levels. An SFN-rich diet (3 µmoles/day SFN versus control chow) decreased the arthritis score in the DMM model of osteoarthritis in the mouse, with a concurrent block of early DMM-induced gene expression changes. CONCLUSION: SFN inhibits the expression of key metalloproteinases implicated in osteoarthritis, independently of Nrf2, and blocks inflammation at the level of NF-κB to protect against cartilage destruction in vitro and in vivo.

Genotype and ripening method affect carotenoid content and bio-accessibility in banana.

Bananas (Musa spp.) are a target crop for provitamin A carotenoids (pVACs) biofortification programs aiming at reducing the negative impact on health caused by vitamin A deficiency in vulnerable populations. However, studies to understand the effect of ripening methods and stages and the genotype on carotenoid content and bioaccessibility in the banana germplasm are scarce. This study evaluated carotenoid content and bioaccessibility in 27 different banana accessions at three maturation stages and two ripening methods (natural ripening and ethylene ripening). Across most accessions, total carotenoid content (TCC) increased from unripe to ripe fruit; only two accessions showed a marginal decrease. The ripening method affected carotenoid accumulation; 18 accessions had lower TCC when naturally ripened compared with the ethylene ripening group, while nine accessions showed higher TCC when ripened with exogenous ethylene, suggesting that treating bananas with exogenous ethylene might directly affect TCC accumulation, but the response is accession dependent. Additionally, carotenoid bioaccessibility varied across genotypes and was correlated with the amount of soluble starch and resistant starch. These findings highlight the importance of ripening methods and genotypes in maximizing banana carotenoid content and bioaccessibility, which could contribute to improving pVACs delivery in biofortification programs.

Unraveling Protein-Metabolite Interactions in Precision Nutrition: A Case Study of Blueberry-Derived Metabolites Using Advanced Computational Methods.

Metabolomics, the study of small-molecule metabolites within biological systems, has become a potent instrument for understanding cellular processes. Despite its profound insights into health, disease, and drug development, identifying the protein partners for metabolites, especially dietary phytochemicals, remains challenging. In the present study, we introduced an innovative in silico, structure-based target prediction approach to efficiently predict protein targets for metabolites. We analyzed 27 blood serum metabolites from nutrition intervention studies' blueberry-rich diets, known for their health benefits, yet with elusive mechanisms of action. Our findings reveal that blueberry-derived metabolites predominantly interact with Carbonic Anhydrase (CA) family proteins, which are crucial in acid-base regulation, respiration, fluid balance, bone metabolism, neurotransmission, and specific aspects of cellular metabolism. Molecular docking showed that these metabolites bind to a common pocket on CA proteins, with binding energies ranging from -5.0 kcal/mol to -9.0 kcal/mol. Further molecular dynamics (MD) simulations confirmed the stable binding of metabolites near the Zn binding site, consistent with known compound interactions. These results highlight the potential health benefits of blueberry metabolites through interaction with CA proteins.

Chronic and postprandial effect of blueberries on cognitive function, alertness, and mood in participants with metabolic syndrome - results from a six-month, double-blind, randomized controlled trial.

BACKGROUND: Anthocyanin and blueberry intakes positively associated with cognitive function in population-based studies and cognitive benefits in randomized controlled trials of adults with self-perceived or clinical cognitive dysfunction. To date, adults with metabolic syndrome (MetS) but without cognitive dysfunction are understudied. OBJECTIVES: Cognitive function, mood, alertness, and sleep quality were assessed as secondary end points in MetS participants, postprandially (>24 h) and following 6-mo blueberry intake. METHODS: A double-blind, randomized controlled trial was conducted, assessing the primary effect of consuming freeze-dried blueberry powder, compared against an isocaloric placebo, on cardiometabolic health >6 mo and a 24 h postprandial period (at baseline). In this secondary analysis of the main study, data from those completing mood, alertness, cognition, and sleep assessments are presented (i.e., n = 115 in the 6 mo study, n = 33 in the postprandial study), using the following: 1) Bond-Lader self-rated scores, 2) electronic cognitive battery (i.e., testing attention, working memory, episodic memory, speed of memory retrieval, executive function, and picture recognition), and 3) the Leeds Sleep Evaluation Questionnaire. Urinary and serum anthocyanin metabolites were quantified, and apolipoprotein E genotype status was determined. RESULTS: Postprandial self-rated calmness significantly improved after 1 cup of blueberries (P = 0.01; q = 0.04; with an 11.6% improvement compared with baseline between 0 and 24 h for the 1 cup group), but all other mood, sleep, and cognitive function parameters were unaffected after postprandial and 6-mo blueberries. Across the ½ and 1 cup groups, microbial metabolites of anthocyanins and chlorogenic acid (i.e., hydroxycinnamic acids, benzoic acids, phenylalanine derivatives, and hippuric acids) and catechin were associated with favorable chronic and postprandial memory, attention, executive function, and calmness. CONCLUSIONS: Although self-rated calmness improved postprandially, and significant cognition-metabolite associations were identified, our data did not support strong cognitive, mood, alertness, or sleep quality improvements in MetS participants after blueberry intervention. This trial was registered at clinicaltrials.gov as NCT02035592.

Gut microbiota and diet matrix modulate the effects of the flavonoid quercetin on atherosclerosis.

Gut bacterial metabolism of dietary flavonoids results in the production of a variety of phenolic acids, whose contributions to health remain poorly understood. Here, we show that supplementation with the commonly consumed flavonoid quercetin impacted gut microbiome composition and resulted in a significant reduction in atherosclerosis burden in conventionally-raised (ConvR) Apolipoprotein E (ApoE) knockout (KO) mice fed a high-MAC (microbiota-accessible carbohydrates) diet. However, this effect was not observed in animals consuming a defined diet containing low levels of MAC. Furthermore, we found that the effect of quercetin on atherosclerosis required gut microbes, as supplementation of this flavonoid to germ-free (GF) ApoE KO mice consuming the high-MAC diet did not affect the development of atherosclerosis. Metabolomic analysis revealed that consumption of quercetin significantly increased plasma levels of benzoylglutamic acid and protocatechuic acid in ConvR mice exposed to the high-MAC diet, while these increases were not observed in GF mice or conventional animals consuming the low-MAC diet supplemented with the flavonoid. Furthermore, levels of these metabolites were negatively associated with atherosclerosis burden. Altogether, these results suggest that the beneficial effects of quercetin on atherosclerosis are influenced by gut microbes and dietary MAC.

A combined DHA-rich fish oil and cocoa flavanols intervention does not improve cognition or brain structure in older adults with memory complaints: results from the CANN randomized, controlled parallel-design study.

BACKGROUND: There is evidence that both omega-3 long-chain polyunsaturated fatty acids (PUFAs) (eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) and cocoa flavanols can improve cognitive performance in both healthy individuals and in those with memory complaints. However, their combined effect is unknown. OBJECTIVES: To investigate the combined effect of EPA/DHA and cocoa flavanols (OM3FLAV) on cognitive performance and brain structures in older adults with memory complaints. METHODS: A randomized placebo-controlled trial of DHA-rich fish oil (providing 1.1 g/d DHA and 0.4 g/d EPA) and a flavanol-rich dark chocolate (providing 500 mg/d flavan-3-ols) was conducted in 259 older adults with either subjective cognitive impairment or mild cognitive impairment. Participants underwent assessment at baseline, 3 mo, and 12 mo. The primary outcome was the number of false-positives on a picture recognition task from the Cognitive Drug Research computerized assessment battery. Secondary outcomes included other cognition and mood outcomes, plasma lipids, brain-derived neurotrophic factor (BDNF), and glucose levels. A subset of 110 participants underwent structural neuroimaging at baseline and at 12 mo. RESULTS: 197 participants completed the study. The combined intervention had no significant effect on any cognitive outcomes, with the exception of reaction time variability (P = 0.007), alertness (P < 0.001), and executive function (P < 0.001), with a decline in function observed in the OM3FLAV group (118.6 [SD 25.3] at baseline versus 113.3 [SD 25.4] at 12 mo for executive function) relative to the control, and an associated decrease in cortical volume (P = 0.039). Compared with the control group, OM3FLAV increased plasma HDL, total cholesterol ratio (P < 0.001), and glucose (P = 0.008) and reduced TG concentrations (P < 0.001) by 3 mo, which were sustained to 12 mo, with no effect on BDNF. Changes in plasma EPA and DHA and urinary flavonoid metabolite concentrations confirmed compliance to the intervention. CONCLUSIONS: These results suggest that cosupplementation with ω-3 PUFAs and cocoa flavanols for 12 mo does not improve cognitive outcomes in those with cognitive impairment. This trial was registered at clinicaltrials.gov as NCT02525198.

Bioavailable Microbial Metabolites of Flavanols Demonstrate Highly Individualized Bioactivity on In Vitro ß-Cell Functions Critical for Metabolic Health.

Dietary flavanols are known for disease preventative properties but are often poorly absorbed. Gut microbiome flavanol metabolites are more bioavailable and may exert protective activities. Using metabolite mixtures extracted from the urine of rats supplemented with flavanols and treated with or without antibiotics, we investigated their effects on INS-1 832/13 ß-cell glucose stimulated insulin secretion (GSIS) capacity. We measured insulin secretion under non-stimulatory (low) and stimulatory (high) glucose levels, insulin secretion fold induction, and total insulin content. We conducted treatment-level comparisons, individual-level dose responses, and a responder vs. non-responder predictive analysis of metabolite composition. While the first two analyses did not elucidate treatment effects, metabolites from 9 of the 28 animals demonstrated significant dose responses, regardless of treatment. Differentiation of responders vs. non-responder revealed that levels of native flavanols and valerolactones approached significance for predicting enhanced GSIS, regardless of treatment. Although treatment-level patterns were not discernable, we conclude that the high inter-individual variability shows that metabolite bioactivity on GSIS capacity is less related to flavanol supplementation or antibiotic treatment and may be more associated with the unique microbiome or metabolome of each animal. These findings suggest flavanol metabolite activities are individualized and point to the need for personalized nutrition practices.

Dietary flavonoids and risk of stroke in women.

BACKGROUND AND PURPOSE: To date, few studies have examined associations between the wide range of flavonoid subclasses and risk of ischemic, hemorrhagic, and total stroke. METHODS: We conducted a prospective study among 69 622 women from the Nurses' Health Study. Total flavonoid and subclass intakes were calculated from semiquantitative food frequency questionnaires collected every 4 years using an updated and extended US Department of Agriculture flavonoid database. RESULTS: During 14 years of follow-up, 1803 incident strokes were confirmed. After adjusting for potential confounders, women in the highest compared with the lowest quintile of flavanone intake had a relative risk of ischemic stroke of 0.81 (95% CI, 0.66-0.99; P=0.04). Citrus fruits/juices, the main dietary source of flavanones, tended to be associated with a reduced risk for ischemic stroke (relative risk, 0.90; 95% CI, 0.77-1.05) comparing extreme quintiles. CONCLUSIONS: Total flavonoid intake was not inversely associated with risk of stroke; however, increased intake of the flavanone subclass was associated with a reduction in the risk of ischemic stroke. Citrus fruit consumption may be associated with a reduction in stroke risk, and experimental data support these epidemiological associations that the flavanone content of citrus fruits may potentially be cardioprotective. Further prospective studies are needed to confirm these associations.

Exploring the Contribution of (Poly)phenols to the Dietary Exposome Using High Resolution Mass Spectrometry Untargeted Metabolomics.

SCOPE: This study presents a workflow to construct a Dietary Exposome Library (DEL) comprised of phytochemicals and their metabolites derived from host and gut microbiome metabolism for use in peak identification/annotation of untargeted metabolomics datasets. METHODS AND RESULTS: An evidence mapping initiative established target analytes related to the consumption of phytochemical-rich foods. Analytes were confirmed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS(n)) analysis of human biospecimens from dietary intervention studies of (poly)phenol-rich diets. One hundred and sixty six verified compounds were subsequently analyzed on an untargeted metabolomics platform to acquire chromatographic and high-resolution mass spectral data for construction of a DEL. The DEL facilitated identification/annotation of 123 metabolites associate with exposure to (poly)phenol enriched diets, which included aromatic ketones, benzoic acids, ellagic acids, caffeoylquinic acids, catecholamines, coumarins, hippuric acid, hydroxytoluenes, phenylamines, stilbenes, urolithins, valerolactones, and xanthonoids, in untargeted metabolomics datasets acquire from human plasma and urine reference materials. CONCLUSIONS: The DEL focusing on (poly)phenols and their metabolites of dietary exposure facilitated identification/annotation of ingested food components and their associated pathways in untargeted metabolomics datasets acquired from human biospecimens. The DEL continues to expand with the aim to provide evidence-based data for dietary metabolites in exposome research and inform the development of dietary intervention strategies.